Pesticidally active proteins and polynucleotides obtainable from Paenibacillus species

ABSTRACT

The subject invention provides unique biological alternatives for pest control. More specifically, the present invention relates to novel pesticidal proteins, novel sources of pesticidal proteins, polynucleotides that encode such toxins, and to methods of using these toxins to control insects and other plant pests. The subject invention relates to the surprising discovery that Paenibacillus species, and proteins therefrom, have toxicity to lepidopterans. There have been no known reports of a Paenibacillus species, strain, or protein having toxicity to lepidopterans. This is also the first known example of a Paenibacillus Cry protein that is toxic to lepidopterans. Furthermore, this is the first known report of Paenibacillus having toxin complex (TC)-like proteins. The DAS 1529  isolate disclosed here is also the first known example of a natural bacterium that produces both a Cry toxin and TC proteins. The subject invention also relates to new classes of Cry and TC proteins that are pesticidally active.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to provisional applicationSerial No. 60/392,633, filed Jun. 28, 2002, and to provisionalapplication Serial No. 60/441,647, filed Jan. 21, 2003.

BACKGROUND OF THE INVENTION

[0002] Insects and other pests cost farmers billions of dollars annuallyin crop losses and in the expense of keeping these pests under control.The losses caused by insect pests in agricultural productionenvironments include decreases in crop yield, reduced crop quality, andincreased harvesting costs. Insect pests are also a burden to vegetableand fruit growers, to producers of ornamental flowers, and to homegardeners and homeowners.

[0003] Cultivation methods, such as crop rotation and the application ofhigh levels of nitrogen fertilizers, have partially addressed problemscaused by agricultural pests. However, economic demands on theutilization of farmland restrict the use of crop rotation. In addition,overwintering traits of some insects are disrupting crop rotations insome areas.

[0004] Thus, synthetic chemical insecticides are relied upon mostheavily to achieve a sufficient level of control. However, the use ofsynthetic chemical insecticides can have several drawbacks. For example,the use of some of these chemicals can adversely affect many beneficialinsects. Target insects have also developed resistance to some chemicalpesticides. This has been partially alleviated by various resistancemanagement strategies, but there is an increasing need for alternativepest control agents. Furthermore, very high populations of larvae, heavyrains, and improper calibration of insecticide application equipment canresult in poor control. The improper use of insecticides raisesenvironmental concerns such as contamination of soil and of both surfaceand underground water supplies. Residues can also remain on treatedfruits, vegetables, and on other treated plants. Working with someinsecticides can also pose hazards to the persons applying them.Therefore, synthetic chemical pesticides are being increasinglyscrutinized for their potential toxic environmental consequences.Stringent new restrictions on the use of pesticides and the eliminationof some effective pesticides from the market place could limiteconomical and effective options for controlling damaging and costlypests.

[0005] Because of the problems associated with the use of syntheticchemical pesticides, there exists a clear need to limit the use of theseagents and a need to identify alternative control agents. Thereplacement of synthetic chemical pesticides, or combination of theseagents with biological pesticides, could reduce the levels of toxicchemicals in the environment.

[0006] Some biological pesticidal agents that are now being used withsome success are derived from the soil microbe Bacillus thuringiensis(B.t.). The soil microbe Bacillus thuringiensis (B.t.) is aGram-positive, spore-forming bacterium. Most strains of B.t. do notexhibit pesticidal activity. Some B.t. strains produce, and can becharacterized by, parasporal crystalline protein inclusions. Theseinclusions often appear microscopically as distinctively shapedcrystals. Some B.t. proteins are highly toxic to pests, such as insects,and are specific in their toxic activity. Certain insecticidal B.t.proteins are associated with the inclusions. These “δ-endotoxins” aredifferent from exotoxins, which have a non-specific host range. Otherspecies of Bacillus also produce pesticidal proteins.

[0007] Certain Bacillus toxin genes have been isolated and sequenced,and recombinant DNA-based products have been produced and approved foruse. In addition, with the use of genetic engineering techniques,various approaches for delivering these toxins to agriculturalenvironments are being perfected. These include the use of plantsgenetically engineered with toxin genes for insect resistance and theuse of stabilized intact microbial cells as toxin delivery vehicles.Thus, isolated Bacillus toxin genes are becoming commercially valuable.

[0008] Commercial use of B.t. pesticides was initially restricted totargeting a narrow range of lepidopteran (caterpillar) pests.Preparations of the spores and crystals of B. thuringiensis subsp.kurstaki have been used for many years as commercial insecticides forlepidopteran pests. For example, B. thuringiensis var. kurstaki HD-1produces a crystalline δ-endotoxin which is toxic to the larvae of anumber of lepidopteran insects.

[0009] More recently, new subspecies of B.t. have been identified, andgenes responsible for active δ-endotoxin proteins have been isolated.Höfte and Whiteley classified B.t. crystal protein genes into four majorclasses (Höfte, H., H. R. Whiteley [1989] Microbiological Reviews52(2):242-255). The classes were CryI (Lepidoptera-specific), CryII(Lepidoptera- and Diptera-specific), CryIII (Coleoptera-specific), andCryIV (Diptera-specific). The discovery of strains specifically toxic toother pests has been reported. For example, CryV and CryVI were proposedto designate a class of toxin genes that are nematode-specific.

[0010] The Lepidopteran-specific CryI crystal proteins, in their naturalstate, are approximately 130- to 140-kDa proteins, which accumulate inbipyramidal crystalline inclusions during the sporulation of B.thuringiensis. These proteins are protoxins which solubilize in thealkaline environment of the insect midgut and are proteolyticallyconverted by crystal-associated or larval-midgut proteases into a toxiccore fragment of 60 to 70 kDa. This activation can also be carried outin vitro with a variety of proteases. The toxic domain is localized inthe N-terminal half of the protoxin. This was demonstrated for CryIA(b)and CryIC proteins through N-terminal amino acid sequencing of thetrypsin-activated toxin. Höfte et al. 1989. Cleavage occurs on theC-terminal end of a conserved region called “Block 5,” thus forming theC-terminus of the core toxin. A short, N-terminal protoxin segment canalso be processed off. The N-terminal cleavage site is also highlyconserved for CryIA and CryID proteins, suggesting that for theseproteins, the N terminus of the toxic fragment is localized at the sameposition. CryIB, however, is different from the other CryI proteins inthis region. It was not known whether this protein is also processed atthe N terminus. Höfte et al. 1989.

[0011] Deletion analysis of several cryI genes further confirmed thatthe 3′ half of the protoxin is not required for toxic activity. One ofthe shortest reported toxic fragments was localized between codons 29and 607 for CryIAb. Further removal of four codons from the 3′ end oreight codons from the 5′ end completely abolished the toxic activity ofthe gene product. Similar observations were made for the cryIA(a) andcryIA(c) genes. Höfte et al. 1989.

[0012] The cryII genes encode 65-kDa proteins which form cuboidalinclusions in strains of several subspecies. These crystal proteins werepreviously designated “P2” proteins, as opposed to the 130-kDa P1crystal proteins present in the same strains. Höfte et al. 1989.

[0013] A cryIIa gene was cloned from B. thuringiensis subsp. kurstakiHD-263 and expressed in Bacillus megaterium. Cells producing the CryIIAprotein were toxic for the lepidopteran species Heliothis virescens andLymantria dispar as well as for larvae of the dipteran Aedes aegypti.Widner and Whitely (1989, J. Bacteriol. 171:965-974) cloned two relatedgenes (cryIIA and cryIIB) from B. thuringiensis subsp. kurstaki HD-1.Both genes encode proteins of 633 amino acids with a predicted molecularmass of 71 kDa (slightly larger than the apparent molecular massdetermined for the P2 proteins produced in B. thuringiensis). Althoughthe CryIIA and CryIIB proteins are highly homologous (˜87% amino acididentity), they differ in their insecticidal spectra. CryIIA is activeagainst both a lepidopteran (Manduca sexta) and a dipteran (Aedesaegypti) species, whereas cryIIB is toxic only to the lepidopteraninsect. Höfte et al. 1989. The CryII toxins, as a group, tend to berelatively more conserved at the sequence level (>80% identical) thanother groups. In contrast, there are many CryI toxins, for example,including some that are less than 60% identical.

[0014] The 1989 nomenclature and classification scheme of Höfte andWhiteley for crystal proteins was based on both the deduced amino acidsequence and the host range of the toxin. That system was adapted tocover 14 different types of toxin genes which were divided into fivemajor classes. The 1989 nomenclature scheme became unworkable as moreand more genes were discovered that encoded proteins with varyingspectrums of pesticidal activity. Thus, a revised nomenclature schemewas adopted, which is based solely on amino acid identity (Crickmore etal., 1998, Microbiology and Molecular Biology Reviews 62:807-813). Themnemonic “cry” has been retained for all of the toxin genes except cytAand cytB, which remain a separate class. Roman numerals have beenexchanged for Arabic numerals in the primary rank, and the parenthesesin the tertiary rank have been removed. Many of the original names havebeen retained, with the noted exceptions, although a number have beenreclassified. There are now at least 37 primary classes of Cry proteins,and two primary classes of cyt toxins. Other types of toxins, such asthose of WO 98/18932 and WO 97/40162, have also been discovered from B.thuringiensis.

[0015] There are some obstacles to the successful agricultural use ofBacillus (and other biological) pesticidal proteins. Certain insects canbe refractory to the effects of Bacillus toxins. Insects such as bollweevils, black cutworm, and Helicoverpa zea, as well as adult insects ofmost species, heretofore have demonstrated no significant sensitivity tomany B.t. δ-endotoxins.

[0016] Another potential obstacle is the development of resistance toB.t. toxins by insects. B.t. protein toxins were initially formulated assprayable insect control agents. A more recent application of B.t.technology has been to isolate and transform plants with genes thatencode these toxins. Transgenic plants subsequently produce the toxins,thereby providing insect control. See U.S. Pat. Nos. 5,380,831;5,567,600; and 5,567,862 to Mycogen Corporation. Transgenic B.t. plantsare quite efficacious, and usage is predicted to be high in some cropsand areas. This has caused some concern that resistance managementissues may arise more quickly than with traditional sprayableapplications. While a number of insects have been selected forresistance to B.t. toxins in the laboratory, only the diamondback moth(Plutella xylostella) has demonstrated resistance in a field setting(Ferre, J. and Van Rie, J., Annu. Rev. Entomol. 47:501-533, 2002).

[0017] Resistance management strategies in B.t. transgene planttechnology have become of great interest (for example, as in a naturalbacterium, multiple diverse toxins can be exposed on the same plant,thereby greatly reducing the chance that an insect that might beresistant to one toxin would survive to spread the resistance). Severalstrategies have been suggested for preserving the ability to effectivelyuse B. thuringiensis toxins. These strategies include high dose withrefuge, and alternation with, or co-deployment of, different toxins(McGaughey et al. (1998), “B.t. Resistance Management,” NatureBiotechnol 16:144-146).

[0018] Thus, there remains a great need for developing additional genesthat can be expressed in plants in order to effectively control variousinsects. In addition to continually trying to discover new B.t. toxins,it would be quite desirable to discover other bacterial sources(distinct from B.t.) that produce toxins that could be used intransgenic plant strategies, or that could be combined with B.t.s toproduce insect-controlling transgenic plants.

[0019] The recent efforts to clone insecticidal toxin genes from thePhotorhabdus/Xenorhabdus group of bacteria present potentialalternatives to toxins derived from B. thuringiensis. It has been knownin the art that bacteria of the genus Xenorhabdus are symbioticallyassociated with the Steinernema nematode. Unfortunately, as reported ina number of articles, the bacteria only had pesticidal activity wheninjected into insect larvae and did not exhibit biological activity whendelivered orally.

[0020] It has been difficult to effectively exploit the insecticidalproperties of the nematode or its bacterial symbiont. Thus, it would bequite desirable to discover proteinaceous agents from Xenorhabdusbacteria that have oral activity so that the products produced therefromcould be formulated as a sprayable insecticide, or the bacterial genesencoding said proteinaceous agents could be isolated and used in theproduction of transgenic plants. WO 95/00647 relates to the use ofXenorhabdus protein toxin to control insects, but it does not recognizeorally active toxins. WO 98/08388 relates to orally administeredpesticidal agents from Xenorhabdus. U.S. Pat. No. 6,048,838 relates toprotein toxins/toxin complexes, having oral activity, obtainable fromXenorhabdus species and strains.

[0021] Photorhabdus and Xenorhabdus spp. are Gram-negative bacteria thatentomopathogenically and symbiotically associate with soil nematodes.These bacteria are found in the gut of entomopathogenic nematodes thatinvade and kill insects. When the nematode invades an insect host, thebacteria are released into the insect haemocoel (the open circulatorysystem), and both the bacteria and the nematode undergo multiple roundsof replication; the insect host typically dies. These bacteria can becultured away from their nematode hosts. For a more detailed discussionof these bacteria, see Forst and Nealson, 60 Microbiol. Rev. 1 (1996),pp. 21-43.

[0022] The genus Xenorhabdus is taxonomically defined as a member of theFamily Enterobacteriaceae, although it has certain traits atypical ofthis family. For example, strains of this genus are typically nitratereduction negative and catalase negative. Xenorhabdus has only recentlybeen subdivided to create a second genus, Photorhabdus, which iscomprised of the single species Photorhabdus luminescens (previouslyXenorhabdus luminescens) (Boemare et al., 1993 Int. J Syst. Bacteriol.43, 249-255). This differentiation is based on several distinguishingcharacteristics easily identifiable by the skilled artisan. Thesedifferences include the following: DNA-DNA characterization studies;phenotypic presence (Photorhabdus) or absence (Xenorhabdus) of catalaseactivity; presence (Photorhabdus) or absence (Xenorhabdus) ofbioluminescence; the Family of the nematode host in that Xenorhabdus isfound in Steinernematidae and Photorhabdus is found inHeterorhabditidae); as well as comparative, cellular fatty-acid analyses(Janse et al. 1990, Lett. Appl. Microbiol. 10, 131-135; Suzuki et al.1990, J. Gen. Appl. Microbiol., 36, 393-401). In addition, recentmolecular studies focused on sequence (Rainey et al. 1995, Int. J. Syst.Bacteriol., 45, 379-381) and restriction analysis (Brunel et al., 1997,App. Environ. Micro., 63, 574-580) of 16S rRNA genes also support theseparation of these two genera.

[0023] The expected traits for Xenorhabdus are the following: Gram stainnegative rods, white to yellow/brown colony pigmentation, presence ofinclusion bodies, absence of catalase, inability to reduce nitrate,absence of bioluminescence, ability to uptake dye from medium, positivegelatin hydrolysis, growth on Enterobacteriaceae selective media, growthtemperature below 37° C., survival under anaerobic conditions, andmotility.

[0024] Currently, the bacterial genus Xenorhabdus is comprised of fourrecognized species, Xenorhabdus nematophilus, Xenorhabdus poinarii,Xenorhabdus bovienii and Xenorhabdus beddingii (Brunel et al., 1997,App. Environ. Micro., 63, 574-580). A variety of related strains havebeen described in the literature (e.g., Akhurst and Boemare 1988 J. Gen.Microbiol., 134, 1835-1845; Boemare et al. 1993 Int. J. Syst. Bacteriol.43, pp. 249-255; Putz et al. 1990, Appl. Environ. Microbiol., 56,181-186, Brunel et al., 1997, App. Environ. Micro., 63, 574-580,Raineyet al. 1995, Int. J. Syst. Bacteriol., 45, 379-381).

[0025] Xenorhabdus and Photorhabdus bacteria secrete a wide variety ofsubstances into the culture medium; these secretions include lipases,proteases, antibiotics and lipopolysaccharides. Purification ofdifferent protease fractions has clearly demonstrated that they are notinvolved in the oral toxic activity of P. luminescens culture medium(which has been subsequently determined to reside with the Tc proteinsonly). Several of these substances have previously been implicated ininsect toxicity but until recently no insecticidal genes had beencloned. However, protease purification and separation will alsofacilitate an examination of their putative role in, for example,inhibiting antibacterial proteins such as cecropin. R. H.ffrench-Constant and Bowen, Current Opinions in Micriobiology, 1999,12:284-288. See R. H. ffrench-Constant et al. 66 AEM No. 8, pp.3310-3329 (August 2000), for a review of various factors involved inPhotorhabdus virulence of insects.

[0026] There has been substantial progress in the cloning of genesencoding insecticidal toxins from both Photorhabdus luminescens andXenorhabdus nematophilus. Toxin-complex encoding genes from P.luminescens were examined first. See, e.g., WO 98/08932. “Parallel”genes were more recently cloned from X. nematophilus. Morgan et al.,Applied and Environmental Microbiology 2001, 67:2062-69.

[0027] Four different toxin complexes (TCs)—Tca, Tcb, Tcc and Tcd—havebeen identified in Photorhabdus spp. Each of these toxin complexesresolves as either a single or dimeric species on a native agarose gelbut resolution on a denaturing gel reveals that each complex consists ofa range of species between 25-280 kDa. The ORFs that encode the TCs fromPhotorhabdus, together with protease cleavage sites (vertical arrows),are illustrated in FIG. 1. See also R. H. ffrench-Constant and Bowen, 57Cell. Mol. Life Sci. 828-833 (2000).

[0028] Genomic libraries of P. luminescens were screened with DNA probesand with monoclonal and/or polyclonal antibodies raised against thetoxins. Four tc loci were cloned: tca, tcb, tcc and tcd. The tca locusis a putative operon of three open reading frames (ORFs), tcaA, tcaB,and tcaC transcribed from the same DNA strand, with a smaller terminalORF (tcaZ) transcribed in the opposite direction. The tcc locus also iscomprised of three ORFs putatively transcribed in the same direction(tccA, tccB, and tccC). The tcb locus is a single large ORF (tcbA), andthe tcd locus is composed of two ORFs (tcdA and tcdB); tcbA and tcdA,each about 7.5 kb, encode large insect toxins. TcdB has some homology toTcaC. Many of these gene products were determined to be cleaved byproteases. For example, both TcbA and TcdA are cleaved into threefragments termed i, ii and iii (e.g. TcbAi, TcbAii and TcbAiii).Products of the tca and tcc ORFs are also cleaved. See FIG. 1. See alsoR. H. ffrench-Constant and D. J. Bowen, Current Opinions inMicrobiology, 1999, 12:284-288.

[0029] Bioassays of the Tca toxin complexes revealed them to be highlytoxic to first instar tomato hornworms (Manduca sexta) when given orally(LD₅₀ of 875 ng per square centimeter of artificial diet). R. H.ffrench-Constant and Bowen 1999. Feeding was inhibited at Tca doses aslow as 40 ng/cm². Given the high predicted molecular weight of Tca, on amolar basis, P. luminescens toxins are highly active and relatively fewmolecules appear to be necessary to exert a toxic effect. R. H.ffrench-Constant and Bowen, Current Opinions in Micriobiology, 1999,12:284-288.

[0030] None of the four loci showed overall similarity to any sequencesof known function in GenBank. Regions of sequence similarity raised somesuggestion that these proteins (TcaC and TccA) may overcome insectimmunity by attacking insect hemocytes. R. H. ffrench-Constant andBowen, Current Opinions in Microbiology, 1999, 12:284-288.

[0031] TcaB, TcbA, and TcdA all show amino acid conservation (˜50%identity), compared with each other, immediately around their predictedprotease cleavage sites. This conservation between three different TCproteins suggests that they may all be processed by the same or similarproteases. TcbA and TcdA also share ˜50% identity overall, as well as asimilar predicted pattern of both carboxy- and amino-terminal cleavage.It was postulated that these proteins might thus be homologs of oneanother. Furthermore, the similar, large size of TcbA and TcdA, and alsothe fact that both toxins appear to act on the gut of the insect, maysuggest similar modes of action. R. H. ffrench-Constant and Bowen,Current Opinions in Microbiology, 1999, 12:284-288.

[0032] Deletion/knock-out studies suggest that products of the tca andtcd loci account for the majority of oral toxicity to lepidopterans.Deletion of either of the tca or tcd genes greatly reduced oral activityagainst Manduca sexta. That is, products of the tca and tcd loci areoral lepidopteran toxins on their own; their combined effect contributedmost of the secreted oral activity. R. H. ffrench-Constant and D. J.Bowen, 57 Cell. Mol. Life. Sci. 831 (2000). Interestingly, deletion ofeither of the tcb or tcc loci alone also reduces mortality, suggestingthat there may be complex interactions among the different geneproducts. Thus, products of the tca locus may enhance the toxicity oftcd products. Alternatively, tcd products may modulate the toxicity oftca products and possibly other complexes. Noting that the above relatesto oral activity against a single insect species, tcb or tcc loci mayproduce toxins that are more active against other groups of insects (oractive via injection directly into the insect haemocoel—the normal routeof delivery when secreted by the bacteria in vivo). R. H.ffrench-Constant and Bowen, Current Opinions in Microbiology, 1999,12:284-288.

[0033] WO 01/11029 discloses nucleotide sequences that encode TcdA andTcbA and have base compositions that have been altered from that of thenative genes to make them more similar to plant genes. Also disclosedare transgenic plants that express Toxin A and Toxin B.

[0034] Of the separate toxins isolated from Photorhabdus luminescens(W-14), those designated Toxin A and Toxin B have been the subject offocused investigation for their activity against target insect speciesof interest (e.g., corn rootworm). Toxin A is comprised of two differentsubunits. The native gene tcdA encodes protoxin TcdA. As determined bymass spectrometry, TcdA is processed by one or more proteases to provideToxin A. More specifically, TcdA is an approximately 282.9 kDa protein(2516 aa) that is processed to provide TcdAi (the first 88 amino acids),TcdAii (the next 1849 aa; an approximately 208.2 kDa protein encoded bynucleotides 265-5811 of tcdA), and TcdAiii, an approximately 63.5 kDa(579 aa) protein (encoded by nucleotides 5812-7551 of tcdA). TcdAii andTcdAiii appear to assemble into a dimer (perhaps aided by TcdAi), andthe dimers assemble into a tetramer of four dimers. Toxin B is similarlyderived from TcbA.

[0035] While the exact molecular interactions of the TC proteins witheach other, and their mechanism(s) of action, are not currentlyunderstood, it is known, for example, that the Tca toxin complex ofPhotorhabdus is toxic to Manduca sexta. In addition, some TC proteinsare known to have “stand alone” insecticidal activity, while other TCproteins are known to potentiate or enhance the activity of thestand-alone toxins. It is known that the TcdA protein is active, alone,against Manduca sexta, but that TcdB and TccC, together, can be used toenhance the activity of TcdA. Waterfield, N. et al., Appl. Environ.Microbiol. 2001, 67:5017-5024. TcbA (there is only one Tcb protein) isanother stand-alone toxin from Photorhabdus. The activity of this toxin(TcbA) can also be enhanced by TcdB together with TccC-like proteins.

[0036] U.S. Patent Application 20020078478 provides nucleotide sequencesfor two potentiator genes, tcdB2 and tccC2, from the tcd genomic regionof Photorhabdus luminescens W-14. It is shown therein that coexpressionof tcdB and tccC1 with tcdA results in enhanced levels of oral insecttoxicity compared to that obtained when tcdA is expressed alone.Coexpression of tcdB and tccC1 with tcdA or tcbA provide enhanced oralinsect activity.

[0037] As indicated in the chart below, TccA has some level of homologywith the N terminus of TcdA, and TccB has some level of homology withthe C terminus of TcdA. TccA and TccB are much less active on certaintest insects than is TcdA. TccA and TccB from Photorhabdus strain W-14are called “Toxin D.” “Toxin A” (TcdA), “Toxin B” (TcbA), and “Toxin C”(TcaA and TcaB) are also indicated below. Furthermore, TcaA has somelevel of homology with TccA and likewise with the N terminus of TcdA.Still further, TcaB has some level of homology with TccB and likewisewith the N terminus of TcdA. TccA and TcaA are of a similar size, as areTccB and TcaB. TcdB has a significant level of similarity (both insequence and size) to TcaC. Photorhabdus strain W14 Some homologyPhotorhabdus nomenclature to: TcaA Toxin C TccA TcaB TccB TcaC TcdB TcbAToxin B TccA Toxin D TcdA N terminus TccB TcdA C terminus TccC TcdAToxin A TccA + TccB TcdB TcaC

[0038] The insect midgut epithelium contains both columnar (structural)and goblet (secretory) cells. Ingestion of tca products by M. sextaleads to apical swelling and blebbing of large cytoplasmic vesicles bythe columnar cells, leading to the eventual extrusion of cell nuclei invesicles into the gut lumen. Goblet cells are also apparently affectedin the same fashion. Products of tca act on the insect midgut followingeither oral delivery or injection. R. H. ffrench-Constant and D. J.Bowen, Current Opinions in Microbiology, 1999, 12:284-288. Purified tcaproducts have shown oral toxicity against Manduca sexta (LD₅₀ of 875ng/cm²). R. H. ffrench-Constant and D. J. Bowen, 57 Cell. Mol. Life Sci.828-833 (2000).

[0039] WO 99/42589 and U.S. Pat. No.6,281,413 disclose TC-like ORFs fromPhotorhabdus luminescens. WO 00/30453 and WO 00/42855 disclose TC-likeproteins from Xenorhabdus. WO 99/03328 and WO 99/54472 (and U.S. Pat.Nos. 6,174,860 and 6,277,823) relate to other toxins from Xenorhabdusand Photorhabdus.

[0040] Relatively recent cloning efforts in Xenorhabdus nematophilusalso appear to have identified novel insecticidal toxin genes withhomology to the P. luminescens tc loci. See, e.g., WO 98/08388 andMorgan et al., Applied and Environmental Microbiology 2001, 67:2062-69.In R. H. ffrench-Constant and D. J. Bowen, Current Opinions inMicrobiology, 1999, 12:284-288, cosmid clones were screened directly fororal toxicity to another lepidopteran, Pieris . One orally toxic cosmidclone was sequenced. Analysis of the sequence in that cosmid suggestedthat there are five different ORF's with similarity to Photorhabdus tcgenes; orf2 and orf5 both have some level of sequence relatedness toboth tcbA and tcdA, whereas orf1 is similar to tccB, orf3 is similar totccC and orf4 is similar to tca C. Importantly, a number of thesepredicted ORFs also share the putative cleavage site documented in P.luminescens, suggesting that active toxins may also be protealyticallyprocessed.

[0041] There are five typical Xenorhabdus TC proteins: XptA1, XptA2,XptB1, XptC1, and XptD1. XptA1 is a “stand-alone” toxin. XptA2 isanother TC protein from Xenorhabdus that has stand-alone toxin activity.See GENBANK Accession No. AJ308438 for sequences from Xenorhabdusnematophilus. XptB1 and XptC1 are the Xenorhabdus potentiators that canenhance the activity of either (or both) of the XptA toxins. XptD1 hassome level of homology with TccB. XptC1 has some level of similarity toTcaC. The XptA2 protein of Xenorhabdus has some degree of similarity tothe TcdA protein. XptB1 has some level of similarity to TccC.

[0042] The finding of somewhat similar, toxin-encoding loci in these twodifferent bacteria is interesting in terms of the possible origins ofthese virulence genes. The X. nematophilus cosmid also appears tocontain transposase-like sequences whose presence may suggest that theseloci can be transferred horizontally between different strains orspecies of bacteria. A range of such transfer events may also explainthe apparently different genomic organization of the tc operons in thetwo different bacteria. Further, only a subset of X. nematophilus and P.luminescens strains appear toxic to M. sexta, suggesting either thatdifferent strains lack the tc genes or that they carry a different tcgene compliment. Detailed analysis of both a strain and toxin phylogenywithin, and between, these bacterial species should help clarify thelikely origin of the toxin genes and how they are maintained indifferent bacterial populations. R. H. ffrench-Constant and Bowen,Current Opinions in Microbiology, 1999, 12:284-288.

[0043] TC proteins and genes have more recently been described fromother insect-associated bacteria such as Serratia entomophila, an insectpathogen. Waterfield et al., TRENDS in Microbiology, Vol. 9, No. 4,April 2001.

[0044] In summary, toxin complex proteins from P. luminescens and X.nematophilus appear to have little homology to previously identifiedbacterial toxins and should provide useful alternatives to toxinsderived from B. thuringiensis. Although they have similar toxic effectson the insect midgut to other orally active toxins, their precise modeof action remains obscure. Future work could clarify their mechanism ofaction.

[0045] Although some Xenorhabdus TC proteins were found to “correspond”(have a similar function and some level of sequence homology) to some ofthe Photorhabdus TC proteins, a given Photorhabdus protein shares onlyabout 40% sequence identity with the “corresponding” Xenorhabdusprotein. This is illustrated below for four “stand-alone” toxins:Identity to P.1. W-14 TcbA Identity to P.1. W-14 TcdA Xwi XptA1 44% 46%Xwi XptA2 41% 41%

[0046] (For a more complete review, see, e.g., Morgan et al., “SequenceAnalysis of Insecticidal Genes from Xenorhabdus nematophiles PMFI296,”Vol. 67, Applied and Environmental Microbiology, May 2001, pp.2062-2069.)

[0047] Bacteria of the genus Paenibacillus are distinguishable fromother bacteria by distinctive rRNA and phenotypic characteristics (C.Ash et al. (1993), “Molecular identification of rRNA group 3 bacilli(Ash, Farrow, Wallbanks and Collins) using a PCR probe test: Proposalfor the creation of a new genus Paenibacillus,” Antonie Van Leeuwenhoek64:253-260). Comparative 16S rRNA sequence analysis demonstrated thatthe genus Bacillus consisted of at least five phyletic lines. RibosomalRNA group 3 bacilli (of Ash, Farrow, Wallbanks, and Collins (1991),comprising Bacillus polymyxa and close relatives), is phylogeneticallyso removed from Bacillus subtilis (the type species of the genus andother aerobic, endospore-forming bacilli) that they were reclassified asa new genus, Paenibacillus.

[0048] Some species in this genus were known to be pathogenic tohoneybees (Paenibacillus Larvae) and scarab beetle grubs (P. popilliaeand P. lentimorbus). Some other Paenibacillus species that have beenfound to be associated with honeybees, but they are non-pathogens. Atleast 18 additional species are known in this genus, including P.thiaminolyticus; they have no known insect association (Shida et al.,1997; Pettersson et al., 1999). Scarabs (coleopterans) are serious pestsof turf, nurseries, and food crops throughout North America, and are ofquarantine concern. See U.S. Department of Agriculture, AgriculturalResearch Service website.

[0049]P. larvae, P. popilliae, and P. lentimorbus are consideredobligate insect pathogens involved with milky disease of scarab beetles(D. P. Stahly et al. (1992), “The genus Bacillus: insect pathogens,” p.1697-1745, In A. Balows et al., ed., The Procaryotes, 2^(nd) Ed., Vol.2, Springer-Verlag, New York, N.Y.). These three Paenibacillus speciesare characteristically slow-growing, fastidious organisms that causedisease by an invasive process in which the bacteria cross the midgutand proliferate to high numbers in the hemolymph and other tissues. Forall three species, some general indications of protein involvement ininsect pathogenicity have been proposed; however, no specific role for aspecific protein has been demonstrated. Stahly et al. concluded for P.larvae that a question of the involvement of a toxin is an open one, andthat the precise cause of death in milky disease (of beetles) is notunderstood.

[0050] A beetle (coleopteran) toxin, Cry18, has been identified instrains of P. popilliae and P. lentimorbus. Cry18 has about 40% identityto Cry2 proteins (Zhang et al., 1997; Harrison et al., 2000). WhileZhang et al. (1997) speculate that Cry18 attacks the midgut tofacilitate entry of vegetative cells to the hemocoel, Harrison et al.note that there is no direct evidence for this role and further statethat “the role, if any, of the paraspore protein in milky disease isunknown.” J. Zhang et al. (1997), “Cloning and Analysis of the First cryGene from Bacillus popilliae,” J. Bacteriol 179:4336-4341; H. Harrisonet al. (2000), “Paenibacillus Associated with Milky Disease in Centraland South American Scarabs,” J. Invertebr. Pathol. 76(3):169-175.

[0051] Stahly et al., Zhang et al., and Harrison et al. all point to thecontrast in evidence for the role of crystal proteins of B.thuringiensis in intoxication of insects (where the high frequency ofinsect symptoms can be explained by the properties of the specificcrystal proteins), versus the case of Paenibacillus and milky disease(where there is no such tie to the effects of a specific toxin).

[0052] Thus, while some species of Paenibacillus were known to bepathogenic to certain coleopterans and some associated with honeybees,no strain of Paenibacillus was heretofore known to be toxic tolepidopterans. Likewise, TC proteins and lepidopteran-toxic Cry proteinshave never been reported in Paenibacillus.

BRIEF SUMMARY OF THE INVENTION

[0053] This is the first known disclosure of Paenibacillus proteintoxins having activity against lepidopteran pests. Some species ofPaenibacillus were known to be insecticidal, but they had activityagainst grubs/beetles/coleopterans. There have been no known reports ofa Paenibacillus species or strain having toxicity to lepidopterans.Thus, the subject invention relates generally to Paenibacillus speciesthat have activity against lepidopterans, and to screening Paenibacillusspp., proteins therefrom, and libraries of clones therefrom for activityagainst lepidopterans.

[0054] More specifically, the subject invention initially stemmed from adiscovery of a novel strain of Paenibacillus referred to herein asDAS1529. This was a surprising discovery for a variety of reasons. Thisstrain produces a unique, lepidopteran-toxic Cry protein. This strain,as well as DB482, produce unique, toxin complex (TC)-like proteins(having some similarity to Xenorhabdus/Photorhabdus TCs). Paenibacillusisolate DB482 and toxins obtainable therefrom are highly preferred, andall are within the scope of the subject invention.

[0055] This is the first known report of Paenibacillus having TC-likeproteins. Thus, the subject invention relates to methods of screeningPaenibacillus spp. for TC-like genes and proteins. Paenibacillus TCproteins of the subject invention are shown herein to be useful toenhance or potentiate the activity of a “stand-alone” Xenorhabdus toxinprotein, for example. TC-like genes identified herein were notheretofore known to exist in the genus Paenibacillus. This discoverybroadens the scope of organisms (bacterial genera) in which TC-likegenes have been found. Thus, the subject invention generally relates toTC-like proteins obtainable from Paenibacillus species, to methods ofscreening Paenibacillus species for such proteins, and the like. Oneexample is Paenibacillus apairius, which was also found to produceTC-like proteins.

[0056] While the subject TC-like proteins have some sequence relatednessto, and characteristics in common with, TC proteins of Xenorhabdus andPhotorabdus, the sequences of the subject TC-like proteins are verydifferent from previously known TC proteins. Thus, the subjectapplication provides new classes of TC-like proteins and genes thatencode these proteins, which are obtainable from bacteria in the generaPaenibacillus, Photorhabdus, Xenorhabdus, and the like.

[0057] Another surprising feature of the DAS1529 strain is that itproduces a unique, B.t.-like Cry protein that is toxic to lepidopterans.The subject Cry toxin is compressed/short and appears to lack a typicalprotoxin portion in its wild-type state. Thus, the subject inventiongenerally relates to screening Paenibacillus isolates forlepidopteran-toxic Cry proteins. The subject invention also relates tomethods of screening Paenibacillus spp. and B. thuringiensis, forexample, for this new class of Cry genes and proteins.

[0058] The DAS1529 strain is the first known example of a naturalbacterium that produces both a Cry-like toxin and TC-like proteins.Further surprising is that this is the first known example of a crytoxin gene being closely associated with (in genetic proximity to) TCprotein genes. These pioneering observations have broad implications andthus enable one skilled in the art to screen appropriate species ofbacteria for these types of unique operons and for these types offurther components of known operons. Such techniques are within thescope of the subject invention.

[0059] A further aspect of the subject invention stems from thesurprising discovery that the DAS1529 strain also produces a solubleinsect toxin that was found to be very similar to a thiaminase. It wassurprising that the Paenibacillus thiaminase protein was found to haveinsecticidal activity. While this type of protein was known, it was inno way expected in the art that this enzyme would have exhibitedtoxin-like activity against insects/insect-like pests. Thus, the subjectinvention also relates to methods of screening Paenibacillus and othersfor insecticidal thiaminase genes and proteins, and to the use of thesegenes and proteins for controlling insects and like pests.

[0060] Other objects, advantages, and features of the subject inventionwill be apparent to one skilled in the art having the benefit of thesubject disclosure.

BRIEF DESCRIPTION OF THE FIGURES

[0061]FIG. 1 shows the TC operons from Photorhabdus.

[0062]FIG. 2 shows a diagram of the DNA from DAS1529 inserted into the“SB12” clone that exhibited pesticidal activity, with open readingframes identified with block and line arrows.

[0063]FIG. 3 shows partial sequence alignments for SEQ ID NO:17 andthiaminase I from Bacillus thiaminolyticus (Campobasso et al., 1998) orAAC44156.

[0064]FIG. 4 shows test results of purified thiaminase from DAS1529 onCEW.

[0065]FIG. 5 shows ORF3-ORF6 in pEt101D.

[0066]FIG. 6 shows Cry1529 (ORF 7) against tobacco bud worm (TBW).

[0067]FIG. 7 shows a comparison/alignment of SEQ ID NO:9 to SEQ ID NO:5(tcaB₂ to tcaB₁); the brackets show the ORF2 junction.

[0068]FIG. 8 shows a phylogenetic tree of DAS1529 ORF7 (Cry1529)compared to other Cry proteins.

[0069]FIGS. 9 and 10 show results of trypsin digestion of wild-type andmodified Cry1529 proteins.

[0070]FIGS. 11A and 11B show sequence alignments for tcaA primer design.

[0071] FIGS. 12A-D show sequence alignments for tcaB primer design.

[0072]FIGS. 13A and 13B show sequence alignments for tcaC primer design.

[0073]FIGS. 14A and 14B show sequence alignments for tccC primer design.

BRIEF DESCRIPTION OF THE SEQUENCES

[0074] SEQ ID NO:1 is the nucleic acid sequence of the entire insert ofSB12.

[0075] SEQ ID NO:2 is the nucleic acid sequence of ORF1, which encodes atcaA-like protein (gene tcaA1, source organism Paenibacillus strainIDAS1529, gene designation tcaA1-1529).

[0076] SEQ ID NO:3 is the amino acid sequence encoded by ORF1.

[0077] SEQ ID NO:4 is the nucleic acid sequence of ORF2, with an ISelement removed, which encodes a tcaB-like protein (gene tcaB1, sourceorganism Paenibacillus strain IDAS 1529, gene designation tcaB1-1529).

[0078] SEQ ID NO:5 is the amino acid sequence encoded by ORF2.

[0079] SEQ ID NO:6 is the nucleic acid sequence of ORF3, which encodes atcaA-like protein (gene tcaA2, source organism Paenibacillus strain IDAS1529, gene designation tcaA2-1529).

[0080] SEQ ID NO:7 is the amino acid sequence encoded by ORF3.

[0081] SEQ ID NO:8 is the nucleic acid sequence of ORF4, which encodes atcaB-like protein (gene tcaB2, source organism Paenibacillus strain IDAS1529, gene designation tcaB2-1529).

[0082] SEQ ID NO:9 is the amino acid sequence encoded by ORF4.

[0083] SEQ ID NO:10 is the nucleic acid sequence of ORF5, which encodesa tcaC-like protein (gene tcaC, source organism Paenibacillus strainIDAS 1529, gene designation tcaC-1529).

[0084] SEQ ID NO:11 is the amino acid sequence encoded by ORF5.

[0085] SEQ ID NO:12 is the nucleic acid sequence of ORF6, which encodesa tccC-like protein.

[0086] SEQ ID NO:13 is the amino acid sequence encoded by ORF6.

[0087] SEQ ID NO:14 is the nucleic acid sequence of ORF7, which encodesa Cry-like protein.

[0088] SEQ ID NO:15 is the amino acid sequence encoded by ORF7.

[0089] SEQ ID NO:16 is the partial nucleic acid sequence of the 16S rDNAof DAS1529 used for taxonomic placement.

[0090] SEQ ID NO:17 is the N-terminal amino acid sequence for thepurified toxin from the broth fraction from DAS1529.

[0091] SEQ ID NO:18 is the amino acid sequence of thiaminase I fromBacillus thiaminolyticus (Campobasso et al., J. Biochem.37(45):15981-15989 (1998)).

[0092] SEQ ID NO:19 is an alternate amino acid sequence encoded by ORF6protein (gene tccC, source organism Paenibacillus strain IDAS 1529, genedesignation tccC-1529).

[0093] SEQ ID NO:20 is gene xptC1, source organism Xenorhabdus strainXwi, gene designation xptC1-Xwi.

[0094] SEQ ID NO:21 is gene xptB1, source organism Xenorhabdus strainXwi, gene designation xptB1-Xwi.

[0095] SEQ ID NO:22 is primer SB101.

[0096] SEQ ID NO:23 is primer SB102.

[0097] SEQ ID NO:24 is primer SB103.

[0098] SEQ ID NO:25 is primer SB104.

[0099] SEQ ID NO:26 is primer SB105.

[0100] SEQ ID NO:27 is primer SB106.

[0101] SEQ ID NO:28 is primer SB212.

[0102] SEQ ID NO:29 is primer SB213.

[0103] SEQ ID NO:30 is primer SB215.

[0104] SEQ ID NO:31 is primer SB217.

[0105] SEQ ID NO:32 is a nucleotide sequence from a tcaA-like gene fromPaenibacillus apairius strain DB482.

[0106] SEQ ID NO:33 is an amino acid sequence from a TcaA-like proteinfrom Paenibacillus apairius strain DB482.

[0107] SEQ ID NO:34 is a nucleotide sequence from a tcaB-like gene fromPaenibacillus apairius strain DB482.

[0108] SEQ ID NO:35 is a nucleotide sequence from a tcaB-like gene fromPaenibacillus apairius strain DB482.

[0109] SEQ ID NO:36 is an amino acid sequence from a TcaB-like proteinfrom Paenibacillus apairius strain DB482.

[0110] SEQ ID NO:37 is an amino acid sequence from a TcaB-like proteinfrom Paenibacillus apairius strain DB482.

[0111] SEQ ID NO:38 is a nucleotide sequence from a tcaC-like gene fromPaenibacillus apairius strain DB482.

[0112] SEQ ID NO:39 is an amino acid sequence from a TcaC-like proteinfrom Paenibacillus apairius strain DB482.

[0113] SEQ ID NO:40 is a nucleotide sequence from a tccC-like gene fromPaenibacillus apairius strain DB482.

[0114] SEQ ID NO:41 is an amino acid sequence from a TccC-like proteinfrom Paenibacillus apairius strain DB482.

[0115] SEQ ID NO:42 is gene tcdB1, source organism Photorhabdus strainW14, gene designation tcdB1-W14.

[0116] SEQ ID NO:43 is gene tcdB2, source organism Photorhabdus strainW14, gene designation tcdB2-W14.

[0117] SEQ ID NO:44 is gene tccC1, source organism Photorhabdus strainW14, gene designation tccC1-W14.

[0118] SEQ ID NO:45 is gene tccC2, source organism Photorhabdus strainW14, gene designation tccC2- W14.

[0119] SEQ ID NO:46 is gene tccC3, source organism Photorhabdus strainW14, gene designation tccC3- W14.

[0120] SEQ ID NO:47 is gene tccC4, source organism Photorhabdus strainW14, gene designation tccC4-W14.

[0121] SEQ ID NO:48 is gene tccC5, source organism Phtorhabdus strainW14, gene designation tccC5-W14.

[0122] SEQ ID NO:49 is the amino acid sequence of the XptA2 TC proteinfrom Xenorhabdus nematophilus Xwi.

DETAILED DESCRIPTION OF THE INVENTION

[0123] The subject invention provides unique biological alternatives forpest control. More specifically, the subject invention provides newsources of proteins that have toxin activity against insects, preferablylepidopterans, and other similar pests. The invention also relates tonew sources of novel polynucleotides that can be used to encode suchtoxins, and to methods of making and methods of using the toxins andcorresponding nucleic acid sequences to control insects and other likeplant pests. The present invention addresses the need for novel insectcontrol agents. The present invention relates to novel pesticidalproteins that are obtainable from Paenibacillus, and other, bacteria.

[0124] The subject invention initially stemmed from a discovery of anovel strain of Paenibacillus. This strain is referred to herein asDAS1529. To demonstrate the broad implications of this discovery, thediscovery of another Paenibacillus strain is also exemplified.

[0125] These strains have been deposited with the Agricultural ResearchService Patent Culture Collection (NRRL) at 1815 North University StreetPeoria, Ill. 61604 U.S.A. The deposited strains and the correspondingdeposit dates and deposit numbers are as follows: Deposited StrainDeposit Date Deposit Number DAS1529 Jun. 19, 2002 NRRL B-30599 DB482Jun. 17, 2003 NRRL B-30670

[0126] These cultures have been deposited for the purposes of thispatent application and were deposited under conditions that assure thataccess to the cultures is available during the pendency of this patentapplication to one determined by the Commissioner of Patents andTrademarks to be entitled thereto under 37 CFR 1.14 and 35 U.S.C. 122.These deposits will be available as required by foreign patent laws incountries wherein counterparts of the subject application, or itsprogeny, are filed. However, it should be understood that theavailability of a deposit does not constitute a license to practice thesubject invention in derogation of patent rights granted by governmentalaction.

[0127] Further, the subject culture deposits were made in accordancewith the provisions of the Budapest Treaty for the Deposit ofMicroorganisms, i.e., they will be stored with all the care necessary tokeep them viable and uncontaminated for a period of at least five yearsafter the most recent request for the furnishing of a sample of thedeposit, and in any case, for a period of at least thirty (30) yearsafter the date of deposit or for the enforceable life of any patentwhich may issue disclosing the culture. The depositor acknowledges theduty to replace the deposit should the depository be unable to furnish asample when requested, due to the condition of the deposit. Allrestrictions on the availability to the public of the subject culturedeposits will be irrevocably removed upon the granting of a patentdisclosing them.

[0128] The discovery of the subject DAS1529 strain was surprising for avariety of reasons. This strain produces a unique, lepidopteran-toxicCry protein. This strain, as well as DB482, also produce unique, toxincomplex (TC)-like proteins (having some similarity toXenorhabdus/Photorhabdus TCs). Paenibacillus isolate DB482 and toxinsobtainable therefrom are highly preferred, and all are within the scopeof the subject invention.

[0129] This is the first known disclosure of a Paenibacillus proteintoxin having activity against a lepidopteran pest. The DAS1529 strainwas found to have toxin activity against lepidopteran pests. This was asurprising discovery. Some species of Paenibacillus were known to haveinsecticidal activity against grubs/beetles/coleopterans. There havebeen no known reports of a Paenibacillus species or strain havingtoxicity to lepidopterans. Thus, the subject invention relates generallyto Paenibacillus species that have activity against lepidopterans, andto screening Paenibacillus cultures, proteins therefrom, and librariesof clones therefrom, for activity against lepidopterans, and/or forgenes that encode “lep toxins,” and more particularly, forlepidopteran-toxic Cry proteins.

[0130] This is also the first known report of Paenibacillus havingTC-like proteins. Thus, the subject invention relates to methods ofscreening Paenibacillus spp. for TC-like genes and proteins. It was verysurprising to find that the DAS1529 and DB482 strains have TC-likeoperons and produce TC proteins (having some level of similarity to TCproteins of Xenorhabdus and Photorhabdus). TC proteins and genesidentified herein were not heretofore known to exist in the genusPaenibacillus. This discovery broadens the scope of organisms (bacterialgenera) in which TC protein genes have been found. Thus, the subjectinvention generally relates to TC proteins obtainable from Paenibacillusspecies, to methods of screening Paenibacillus species for suchproteins, and the like. An example of a Paenibacillus species foundusing the methods of the subject invention is Paenibacillus apairiusstrain DB482. This P. apairius strain also produces unique TC-likeproteins.

[0131] While the subject TC proteins have some characteristics in commonwith TC proteins of Xenorhabdus and Photorabdus, the subject TC proteinsare unique and different from previously known TC proteins. Thus, thesubject application provides new classes of TC-like proteins and genesthat encode these proteins obtainable from bacteria in the generaPaenibacillus, Photorhabdus, Xenorhabdus, Serratia, and the like.

[0132] The subject invention also relates to lepidopteran-toxic Cryproteins that are obtainable from Paenibacillus species. Thus, thesubject invention relates to methods of screening Paenibacillus speciesfor cry genes and Cry proteins that have toxin activity against alepidopteran pest.

[0133] The DAS1529 Cry toxin is a very unique, B.t.-like Cry proteintoxin. One other strain of Paenibacillus, a strain with activity againstgrubs, was known to produce a coleopteran-toxic Cry protein. That was aCry18 protein, which was most related to Cry2 proteins (but only about40% identity). The Cry protein exemplified herein shows only a low levelof sequence identity and similarity to previously known Cry proteins.With that noted, of all the known B.t. Cry proteins, the subject Cryprotein shares the most similarity to Cry1 proteins. One surprisingaspect of the subject Cry protein is that it is very short, i.e., evenshorter than the Cry1Fa core toxin. The subject Cry protein has anidentifiable Block 5 region at or near its C terminus. This toxin in itswild-type state has no protoxin portion, which is typically found onCry1 toxins. The subject Cry toxin is surprisingly compressed. Thus, thesubject invention generally relates to a new class of Cry proteins. Thisdisclosure is also significant to the search for additional cry genesfrom Bacillus thuringiensis (B.t.). As would be clear to one skilled inthe art having the benefit of the subject disclosure, other bacteria,such as B.t. and other Bacillus spp. (including sphaericus) could bescreened for similar toxins and toxin genes. These methods of screeningare within the scope of the subject invention.

[0134] The DAS1529 strain is the first known example of a naturalbacterium that produces both a Cry-like toxin and TC-like proteins.Further surprising is that this is the first known example of a crytoxin gene being closely associated with (in genetic proximity to) TCprotein genes. These pioneering observations thus enable one skilled inthe art to screen appropriate species of bacteria for these types ofunique operons and for these types of further components of knownoperons. Such techniques are within the scope of the subject invention.The DAS1529 strain is an interesting example of a wild type strainhaving a TC-like operon with multiple TC protein genes of the samegeneral type (i.e., in this case, two tcaA-like and two tcaB-likegenes). This could have implications for further gene discovery.

[0135] A further aspect of the subject invention stems from thesurprising discovery that the Paenibacillus thiaminase protein hasinsecticidal activity. While this protein was known, it was in no wayexpected in the art that this enzyme would have exhibited toxin-likeactivity against insects/insect-like pests.

Paenibacillus TC Proteins

[0136] More specifically regarding the exemplified TC proteins, thefollowing TC proteins from strain DAS1529 have been fully characterizedherein: two TcaA-like proteins (TcaA₁ and TcaA₂), two TcaB-like proteins(TcaB₁ and TcaB₂), a TcaC protein, and a TccC-like protein. The TcaA₁and TcaA₂ proteins are highly similar to each other at the sequencelevel, and the tcaB₁ and tcaB₂ proteins are highly similar to each otherat the sequence level. TC-like proteins obtainable from Paenibacillusapairius are also exemplified herein, and are within the scope of thesubject invention.

[0137] The TC proteins of the subject invention can be used like otherTC proteins. This would be readily apparent to one skilled in the arthaving the benefit of the subject disclosure when viewed in light ofwhat was known in the art. See, e.g., the Background section, above,which discusses R. H. ffrench-Constant and Bowen (2000) and U.S. Pat.No. 6,048,838. For example, it was known that the Tca toxin complex ofPhotorhabdus is highly toxic to Manduca sexta.

[0138] While the exact molecular interactions of the TC proteins witheach other, and their mechanism(s) of action, are not currentlyunderstood, some TC proteins were known to have “stand alone”insecticidal activity, and other TC proteins were known to enhance theactivity of the stand-alone toxins produced by the same given organism.For example, it was known that the TcdA protein was active againstManduca sexta. TcaC and TccC, together, can be used to enhance theactivity of TcdA. TcdB can be used (in place of TcaC) with TccC as apotentiator. TcbA is another Photorhabdus TC protein with stand-alonetoxin activity. TcaC (or TcdB) together with TccC can also be used toenhance/potentiate the toxin activity of TcbA.

[0139] Photorhabdus TC proteins and “corresponding” TC proteins/genesfrom Paenibacillus are summarized below. Photorhabdus strain W14Photorhabdus Paenibacillus Photorhabdus nomenclature Self homology 1529TcaA Toxin C TccA ORF3 (& 1) TcaB TccB ORF4 (& 2) TcaC TcdB ORF5 TcbToxin B TccA Toxin D TcdA N terminus TccB TcdA C terminus TccC ORF6 TcdAToxin A TccA + TccB TcdB TcaC

[0140] As indicated above, TccA has some level of homology with the Nterminus of TcdA, and TccB has some level of homology with the Cterminus of TcdA. Furthermore, TcdA is about 280 kDa, and TccA togetherwith TccB are of about the same size, if combined, as TcdA. Furthermore,TcaA has some level of homology with TccA and likewise with the Nterminus of TcdA. Still further, TcaB has some level of homology withTccB and likewise with the N terminus of TcdA. TccA and TcaA are of asimilar size, as are TccB and TcaB.

[0141] Although some Xenorhabdus TC proteins were found to “correspond”to some of the Photorhabdus TC proteins, the “corresponding” proteinsshare only about 40% (approximately) sequence identity with each other.The subject TC proteins from Paenibacillus have about that same degreeof sequence relatedness (˜40% identity) with prior TC proteins.

[0142] As described in more detail below, one or more toxins of thesubject invention can be used in combination with each other and/or withother toxins (i.e., the Photorhabdus Tca complex was known to be activeagainst Manduca sexta; various “combinations” of Photorhabdus TCproteins, for example, can be used together to enhance the activity ofother, stand-alone Photorhabdus toxins; the use of Photorhabdus toxins“with” B.t. toxins, for example, has been proposed for resistancemanagement.) Furthermore, Paenibacillus TC proteins of the subjectinvention are shown herein to be useful to enhance or potentiate theactivity of a “stand-alone” Xenorhabdus toxin protein, for example.Provisional application No. 60/441,723 (Timothy D. Hey et al.), entitled“Mixing and Matching TC Proteins for Pest Control,” relates to thesurprising discovery that a TC protein derived from an organism of onegenus such as Photorhabdus can be used interchangeably with a“corresponding” TC protein derived from an organism of another genus.Further surprising data along these lines is presented below whichfurther illustrate the utility of the Paenibacillus TC proteins of thesubject invention. One reason that these results might be surprising isthat there is only ˜40% sequence identity between “corresponding”Xhenorhabdus, Photorhabdus, and the subject Paenibacillus TC proteins.

[0143] Proteins and toxins. The present invention provides easilyadministered, functional proteins. The present invention also provides amethod for delivering insecticidal toxins that are functionally activeand effective against many orders of insects, preferably lepidopteraninsects. By “functional activity” (or “active against”) it is meantherein that the protein toxins function as orally active insect controlagents (alone or in combination with other proteins), that the proteinshave a toxic effect (alone or in combination with other proteins), orare able to disrupt or deter insect growth and/or feeding which may ormay not cause death of the insect. When an insect comes into contactwith an effective amount of a “toxin” of the subject invention deliveredvia transgenic plant expression, formulated protein composition(s),sprayable protein composition(s), a bait matrix or other deliverysystem, the results are typically death of the insect, inhibition of thegrowth and/or proliferation of the insect, and/or prevention of theinsects from feeding upon the source (preferably a transgenic plant)that makes the toxins available to the insects. Functional proteins ofthe subject invention can also enhance or improve the activity of othertoxin proteins. Thus, terms such as “toxic,” “toxicity,” “toxinactivity,” and “pesticidally active” as used herein are meant to conveythat the subject “toxins” have “functional activity” as defined herein.

[0144] Complete lethality to feeding insects is preferred, but is notrequired to achieve functional activity. If an insect avoids the toxinor ceases feeding, that avoidance will be useful in some applications,even if the effects are sublethal or lethality is delayed or indirect.For example, if insect resistant transgenic plants are desired, thereluctance of insects to feed on the plants is as useful as lethaltoxicity to the insects because the ultimate objective is avoidinginsect-induced plant damage.

[0145] There are many other ways in which toxins can be incorporatedinto an insect's diet. For example, it is possible to adulterate thelarval food source with the toxic protein by spraying the food with aprotein solution, as disclosed herein. Alternatively, the purifiedprotein could be genetically engineered into an otherwise harmlessbacterium, which could then be grown in culture, and either applied tothe food source or allowed to reside in the soil in an area in whichinsect eradication was desirable. Also, the protein could be geneticallyengineered directly into an insect food source. For instance, the majorfood source for many insect larvae is plant material. Therefore thegenes encoding toxins can be transferred to plant material so that saidplant material expresses the toxin of interest.

[0146] Transfer of the functional activity to plant or bacterial systemstypically requires nucleic acid sequences, encoding the amino acidsequences for the toxins, integrated into a protein expression vectorappropriate to the host in which the vector will reside. One way toobtain a nucleic acid sequence encoding a protein with functionalactivity is to isolate the native genetic material from the bacterialspecies which produce the toxins, using information deduced from thetoxin's amino acid sequence, as disclosed herein. The native sequencescan be optimized for expression in plants, for example, as discussed inmore detail below. Optimized polynucleotide can also be designed basedon the protein sequence.

[0147] The subject invention provides new classes of toxins havingadvantageous pesticidal activities. One way to characterize theseclasses of toxins and the polynucleotides that encode them is bydefining a polynucleotide by its ability to hybridize, under a range ofspecified conditions, with an exemplified nucleotide sequence (thecomplement thereof and/or a probe or probes derived from either strand)and/or by their ability to be amplified by PCR using primers derivedfrom the exemplified sequences.

[0148] There are a number of methods for obtaining the pesticidal toxinsof the instant invention. For example, antibodies to the pesticidaltoxins disclosed and claimed herein can be used to identify and isolateother toxins from a mixture of proteins. Specifically, antibodies may beraised to the portions of the toxins which are most constant and mostdistinct from other toxins. These antibodies can then be used tospecifically identify equivalent toxins with the characteristic activityby immunoprecipitation, enzyme linked immunosorbent assay (ELISA), orwestern blotting. Antibodies to the toxins disclosed herein, or toequivalent toxins, or to fragments of these toxins, can be readilyprepared using standard procedures. Monoclonal, polyclonal, specific,and/or cross-reactive antibodies can be made and used according to thesubject invention. Such antibodies can be included in test kits fordetecting the presence of proteins (and antigenic fragments thereof) ofthe subject invention.

[0149] One skilled in the art would readily recognize that toxins (andgenes) of the subject invention can be obtained from a variety ofsources. A toxin “from” or “obtainable from” the subject DAS 1529isolate and/or the P. apiarius isolate means that the toxin (or asimilar toxin) can be obtained from this isolate or some other source,such as another bacterial strain or a transgenic plant. For example, oneskilled in the art will readily recognize that, given the disclosure ofa bacterial gene and toxin, a plant can be engineered to produce thetoxin. Antibody preparations, nucleic acid probes (DNA and RNA), and thelike may be prepared using the polynucleotide and/or amino acidsequences disclosed herein and used to screen and recover other toxingenes from other (natural) sources. Toxins of the subject invention canbe obtained from a variety of sources/source microorganisms.

[0150] Polynucleotides and probes. The subject invention furtherprovides nucleotide sequences that encode the toxins of the subjectinvention. The subject invention further provides methods of identifyingand characterizing genes that encode pesticidal toxins. In oneembodiment, the subject invention provides unique nucleotide sequencesthat are useful as hybridization probes and/or primers for PCRtechniques. The primers produce characteristic gene fragments that canbe used in the identification, characterization, and/or isolation ofspecific toxin genes. The nucleotide sequences of the subject inventionencode toxins that are distinct from previously described toxins.

[0151] The polynucleotides of the subject invention can be used to formcomplete “genes” to encode proteins or peptides in a desired host cell.For example, as the skilled artisan would readily recognize, the subjectpolynucleotides can be appropriately placed under the control of apromoter in a host of interest, as is readily known in the art.

[0152] As the skilled artisan knows, DNA typically exists in adouble-stranded form. In this arrangement, one strand is complementaryto the other strand and vice versa. As DNA is replicated in a plant (forexample), additional complementary strands of DNA are produced. The“coding strand” is often used in the art to refer to the strand thatbinds with the anti-sense strand. The mRNA is transcribed from the“anti-sense” strand of DNA. The “sense” or “coding” strand has a seriesof codons (a codon is three nucleotides that can be read as athree-residue unit to specify a particular amino acid) that can be readas an open reading frame (ORF) to form a protein or peptide of interest.In order to express a protein in vivo, a strand of DNA is typicallytranscribed into a complementary strand of mRNA which is used as thetemplate for the protein. Thus, the subject invention includes the useof the exemplified polynucleotides shown in the attached sequencelisting and/or equivalents including the complementary strands. RNA andPNA (peptide nucleic acids) that are functionally equivalent to theexemplified DNA are included in the subject invention.

[0153] In one embodiment of the subject invention, bacterial isolatescan be cultivated under conditions resulting in high multiplication ofthe microbe. After treating the microbe to provide single-strandedgenomic nucleic acid, the DNA can be contacted with the primers of theinvention and subjected to PCR amplification. Characteristic fragmentsof toxin-encoding genes will be amplified by the procedure, thusidentifying the presence of the toxin-encoding gene(s).

[0154] Further aspects of the subject invention include genes andisolates identified using the methods and nucleotide sequences disclosedherein. The genes thus identified encode toxins active against pests.

[0155] Toxins and genes of the subject invention can be identified andobtained by using oligonucleotide probes, for example. These probes aredetectable nucleotide sequences which may be detectable by virtue of anappropriate label or may be made inherently fluorescent as described inInternational Application No. WO 93/16094. The probes (and thepolynucleotides of the subject invention) may be DNA, RNA, or PNA. Inaddition to adenine (A), cytosine (C), guanine (G), thymine (T), anduracil (U; for RNA molecules), synthetic probes (and polynucleotides) ofthe subject invention can also have inosine (a neutral base capable ofpairing with all four bases; sometimes used in place of a mixture of allfour bases in synthetic probes). Thus, where a synthetic, degenerateoligonucleotide is referred to herein, and “n” is used generically, “n”can be G, A, T, C, or inosine. Ambiguity codes as used herein are inaccordance with standard IUPAC naming conventions as of the filing ofthe subject application (for example, R means A or G, Y means C or T,etc.).

[0156] As is well known in the art, if a probe molecule hybridizes witha nucleic acid sample, it can be reasonably assumed that the probe andsample have substantial homology/similarity/identity. Preferably,hybridization of the polynucleotide is first conducted followed bywashes under conditions of low, moderate, or high stringency bytechniques well-known in the art, as described in, for example, Keller,G. H., M. M. Manak (1987) DNA Probes, Stockton Press, New York, N.Y.,pp. 169-170. For example, as stated therein, low stringency conditionscan be achieved by first washing with 2× SSC (Standard SalineCitrate)/0.1% SDS (Sodium Dodecyl Sulfate) for 15 minutes at roomtemperature. Two washes are typically performed. Higher stringency canthen be achieved by lowering the salt concentration and/or by raisingthe temperature. For example, the wash described above can be followedby two washings with 0.1× SSC/0.1% SDS for 15 minutes each at roomtemperature followed by subsequent washes with 0.1×SSC/0.1% SDS for 30minutes each at 55° C. These temperatures can be used with otherhybridization and wash protocols set forth herein and as would be knownto one skilled in the art (SSPE can be used as the salt instead of SSC,for example). The 2× SSC/0.1% SDS can be prepared by adding 50 ml of 20×SSC and 5 ml of 10% SDS to 445 ml of water. 20× SSC can be prepared bycombining NaCl (175.3 g/0.150 M), sodium citrate (88.2 g/0.015 M), andwater to 1 liter, followed by adjusting pH to 7.0 with 10 N NaOH. 10%SDS can be prepared by dissolving 10 g of SDS in 50 ml of autoclavedwater, diluting to 100 ml, and aliquotting.

[0157] Detection of the probe provides a means for determining in aknown manner whether hybridization has been maintained. Such a probeanalysis provides a rapid method for identifying toxin-encoding genes ofthe subject invention. The nucleotide segments which are used as probesaccording to the invention can be synthesized using a DNA synthesizerand standard procedures. These nucleotide sequences can also be used asPCR primers to amplify genes of the subject invention.

[0158] Probes for use according to the subject invention can be derivedfrom a variety of sources, such as any gene mentioned or suggestedherein. For example, all or part of any of the following types of genes(coding and/or noncoding or complementary strands thereof) can be usedaccording to the subject invention: tcaA, tcaB, tcaC, tcbA, tccA, tccB,tccC, tcdA, tcdB, xptA1, xptD1, xptB1, xptC1, xptA2, sepA, sepB, andsepC. Unless specifically indicated otherwise, reference to a “tccC”gene, for example, includes all specific alleles (such as tccC1 andtccC2) of this type of gene. The same is true for all the other genes(e.g., tcdB2, tccC3, and the alleles mentioned in Table 17).

[0159] Hybridization characteristics of a molecule can be used to definepolynucleotides of the subject invention. Thus the subject inventionincludes polynucleotides (and/or their complements, preferably theirfull complements) that hybridize with a polynucleotide (or anoligonucleotide or primer) exemplified or suggested herein.

[0160] As used herein “stringent” conditions for hybridization refers toconditions which achieve the same, or about the same, degree ofspecificity of hybridization as the conditions employed by the currentapplicants. Specifically, hybridization of immobilized DNA on Southernblots with ³²P-labeled gene-specific probes was performed by standardmethods (see, e.g., Maniatis, T., E. F. Fritsch, J. Sambrook [1982]Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory,Cold Spring Harbor, N.Y.). In general, hybridization and subsequentwashes were carried out under conditions that allowed for detection oftarget sequences. For double-stranded DNA gene probes, hybridization wascarried out overnight at 20-25° C. below the melting temperature (Tm) ofthe DNA hybrid in 6× SSPE, 5× Denhardt's solution, 0.1% SDS, 0.1 mg/mldenatured DNA. The melting temperature is described by the followingformula (Beltz, G. A., K. A. Jacobs, T. H. Eickbush, P. T. Cherbas, andF. C. Kafatos [1983] Methods of Enzymology, R. Wu, L. Grossman and K.Moldave [eds.] Academic Press, New York 100:266-285):

Tm=81.5° C.+16.6 Log[Na+]+0.41(%G+C)−0.61(%formamide)−600/length ofduplex in base pairs.

[0161] Washes are typically carried out as follows:

[0162] (1) Twice at room temperature for 15 minutes in 1× SSPE, 0.1% SDS(low stringency wash).

[0163] (2) Once at Tm-20° C. for 15 minutes in 0.2× SSPE, 0.1% SDS(moderate stringency wash).

[0164] For oligonucleotide probes, hybridization was carried outovernight at 10-20° C. below the melting temperature (Tm) of the hybridin 6× SSPE, 5× Denhardt's solution, 0.1% SDS, 0.1 mg/ml denatured DNA.Tm for oligonucleotide probes was determined by the following formula:

Tm(° C.)=2(number T/A base pairs)+4(number G/C base pairs)

[0165] (Suggs, S. V., T. Miyake, E. H. Kawashime, M. J. Johnson, K.Itakura, and R. B. Wallace [1981] ICN-UCLA Symp. Dev. Biol. UsingPurified Genes, D. D. Brown [ed.], Academic Press, New York,23:683-693).

[0166] Washes were typically carried out as follows:

[0167] (1) Twice at room temperature for 15 minutes 1× SSPE, 0.1% SDS(low stringency wash).

[0168] (2) Once at the hybridization temperature for 15 minutes in 1×SSPE, 0.1% SDS (moderate stringency wash).

[0169] In general, salt and/or temperature can be altered to changestringency. With a labeled DNA fragment >70 or so bases in length, thefollowing conditions can be used: Low: 1 or 2x SSPE, room temperatureLow: 1 or 2x SSPE, 42° C. Moderate: 0.2x or 1x SSPE, 65° C. High: 0.1xSSPE, 65° C.

[0170] Duplex formation and stability depend on substantialcomplementarity between the two strands of a hybrid, and, as notedabove, a certain degree of mismatch can be tolerated. Therefore, theprobe sequences of the subject invention include mutations (both singleand multiple), deletions, insertions of the described sequences, andcombinations thereof, wherein said mutations, insertions and deletionspermit formation of stable hybrids with the target polynucleotide ofinterest. Mutations, insertions, and deletions can be produced in agiven polynucleotide sequence in many ways, and these methods are knownto an ordinarily skilled artisan. Other methods may become known in thefuture.

[0171] PCR technology. Polymerase Chain Reaction (PCR) is a repetitive,enzymatic, primed synthesis of a nucleic acid sequence. This procedureis well-known and commonly used by those skilled in this art (seeMullis, U.S. Pat. Nos. 4,683,195, 4,683,202, and 4,800,159; Saiki,Randall K., Stephen Scharf, Fred Faloona, Kary B. Mullis, Glenn T. Horn,Henry A. Erlich, Norman Arnheim [1985] “Enzymatic Amplification ofβ-Globin Genomic Sequences and Restriction Site Analysis for Diagnosisof Sickle Cell Anemia,” Science 230:1350-1354). PCR is based on theenzymatic amplification of a DNA fragment of interest that is flanked bytwo oligonucleotide primers that hybridize to opposite strands of thetarget sequence. The primers are oriented with the 3′ ends pointingtowards each other. Repeated cycles of heat denaturation of thetemplate, annealing of the primers to their complementary sequences, andextension of the annealed primers with a DNA polymerase result in theamplification of the segment defined by the 5′ ends of the PCR primers.The extension product of each primer can serve as a template for theother primer, so each cycle essentially doubles the amount of DNAfragment produced in the previous cycle. This results in the exponentialaccumulation of the specific target fragment, up to several million-foldin a few hours. By using a thermostable DNA polymerase such as Taqpolymerase, isolated from the thermophilic bacterium Thermus aquaticus,the amplification process can be completely automated. Other enzymeswhich can be used are known to those skilled in the art.

[0172] The DNA sequences of the subject invention can be used as primersfor PCR amplification. In performing PCR amplification, a certain degreeof mismatch can be tolerated between primer and template. Therefore,mutations, deletions, and insertions (especially additions ofnucleotides to the 5′ end) of the exemplified primers fall within thescope of the subject invention. Mutations, insertions, and deletions canbe produced in a given primer by methods known to an ordinarily skilledartisan.

[0173] Modification of genes and toxins. The genes and toxins usefulaccording to the subject invention include not only the specificallyexemplified full-length sequences, but also portions, segments and/orfragments (including internal and/or terminal deletions compared to thefull-length molecules) of these sequences, variants, mutants, chimerics,and fusions thereof. For example, toxins of the subject invention may beused in the form of chimeric toxins produced by combining portions oftwo or more toxins/proteins.

[0174] Proteins of the subject invention can have substituted aminoacids so long as they retain the characteristic pesticidal/functionalactivity of the proteins specifically exemplified herein. “Variant”genes have nucleotide sequences that encode the same toxins orequivalent toxins having pesticidal activity equivalent to anexemplified protein. The terms “variant proteins” and “equivalenttoxins” refer to toxins having the same or essentially the samebiological/functional activity against the target pests and equivalentsequences as the exemplified toxins. As used herein, reference to an“equivalent” sequence refers to sequences having amino acidsubstitutions, deletions, additions, or insertions which improve or donot adversely affect pesticidal activity. Fragments retaining pesticidalactivity are also included in this definition. Fragments and otherequivalents that retain the same or similar function, or “toxinactivity,” of a corresponding fragment of an exemplified toxin arewithin the scope of the subject invention. Changes, such as amino acidsubstitutions or additions, can be made for a variety of purposes, suchas increasing (or decreasing) protease stability of the protein (withoutmaterially/substantially decreasing the functional activity of thetoxin).

[0175] Equivalent toxins and/or genes encoding these equivalent toxinscan be obtained/derived from wild-type or recombinant bacteria and/orfrom other wild-type or recombinant organisms using the teachingsprovided herein. Other Bacillus, Paenibacillus, Photorhabdus, andXenorhabdus species, for example, can be used as source isolates.

[0176] Variations of genes may be readily constructed using standardtechniques for making point mutations, for example. In addition, U.S.Pat. No. 5,605,793, for example, describes methods for generatingadditional molecular diversity by using DNA reassembly after randomfragmentation. Variant genes can be used to produce variant proteins;recombinant hosts can be used to produce the variant proteins. Usingthese “gene shuffling” techniques, equivalent genes and proteins can beconstructed that comprise any 5, 10, or 20 contiguous residues (aminoacid or nucleotide) of any sequence exemplified herein. As one skilledin the art knows, the gene shuffling techniques can be adjusted toobtain equivalents having, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64,65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82,83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100,101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114,115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128,129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142,143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156,157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170,171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184,185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198,199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212,213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226,227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240,241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254,255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268,269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282,283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296,297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310,311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324,325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338,339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352,353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366,367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380,381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394,395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408,409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422,423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436,437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450,451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464,465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478,479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492,493, 494, 495, 496, 497, 498, 499, or 500 or more contiguous residues(amino acid or nucleotide), corresponding to a segment (of the samesize) in any of the exemplified sequences (or the complements (fullcomplements) thereof). Similarly sized segments, especially those forconserved regions, can also be used as probes and/or primers.

[0177] Fragments of full-length genes can be made using commerciallyavailable exonucleases or endonucleases according to standardprocedures. For example, enzymes such as Bal31 or site-directedmutagenesis can be used to systematically cut off nucleotides from theends of these genes. Also, genes which encode active fragments may beobtained using a variety of restriction enzymes. Proteases may be usedto directly obtain active fragments of these toxins.

[0178] It is within the scope of the invention as disclosed herein thattoxins may be truncated and still retain functional activity. By“truncated toxin” is meant that a portion of a toxin protein may becleaved and yet still exhibit activity after cleavage. Cleavage can beachieved by proteases inside or outside of the insect gut. Furthermore,effectively cleaved proteins can be produced using molecular biologytechniques wherein the DNA bases encoding said toxin are removed eitherthrough digestion with restriction endonucleases or other techniquesavailable to the skilled artisan. After truncation, said proteins can beexpressed in heterologous systems such as E. coli, baculoviruses,plant-based viral systems, yeast and the like and then placed in insectassays as disclosed herein to determine activity. It is well-known inthe art that truncated toxins can be successfully produced so that theyretain functional activity while having less than the entire,full-length sequence. It is well known in the art that B.t. toxins canbe used in a truncated (core toxin) form. See, e.g., Adang et al., Gene36:289-300 (1985), “Characterized full-length and truncated plasmidclones of the crystal protein of Bacillus thuringiensis subsp kurstakiHD-73 and their toxicity to Manduca sexta.” There are other examples oftruncated proteins that retain insecticidal activity, including theinsect juvenile hormone esterase (U.S. Pat. No. 5,674,485 to the Regentsof the University of California). As used herein, the term “toxin” isalso meant to include functionally active truncations. On the otherhand, a protoxin portion (typically the C-terminal half of a typicalB.t. Cry toxin) can be added to form an active, full-length protein.See, e.g., U.S. Pat. No.6,218,188.

[0179] Certain toxins of the subject invention have been specificallyexemplified herein. As these toxins are merely exemplary of the toxinsof the subject invention, it should be readily apparent that the subjectinvention comprises variant or equivalent toxins (and nucleotidesequences coding for equivalent toxins) having the same or similarpesticidal activity of the exemplified toxin. Equivalent toxins willhave amino acid similarity (and/or homology) with an exemplified toxin.The amino acid identity will typically be greater than 60%, preferablygreater than 75%, more preferably greater than 80%, even more preferablygreater than 90%, and can be greater than 95%. Preferred polynucleotidesand proteins of the subject invention can also be defined in terms ofmore particular identity and/or similarity ranges. For example, theidentity and/or similarity can be 49, 50, 51, 52, 53, 54, 55, 56, 57,58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75,76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93,94, 95, 96, 97, 98, or 99% as compared to a sequence exemplified herein.Unless otherwise specified, as used herein percent sequence identityand/or similarity of two nucleic acids is determined using the algorithmof Karlin and Altschul (1990), Proc. Natl. Acad. Sci. USA 87:2264-2268,modified as in Karlin and Altschul (1993), Proc. Natl. Acad. Sci. USA90:5873-5877. Such an algorithm is incorporated into the NBLAST andXBLAST programs of Altschul et al. (1990), J. Mol. Biol. 215:402-410.BLAST nucleotide searches are performed with the NBLAST program,score=100, wordlength=12. To obtain gapped alignments for comparisonpurposes, Gapped BLAST is used as described in Altschul et al. (1997),Nucl. Acids Res. 25:3389-3402. When utilizing BLAST and Gapped BLASTprograms, the default parameters of the respective programs (NBLAST andXBLAST) are used. See NCBI/NIH website. The scores can also becalculated using the methods and algorithms of Crickmore et al. asdescribed in the Background section, above.

[0180] The amino acid homology/similarity/identity will be highest incritical regions of the toxin which account for biological activity orare involved in the determination of three-dimensional configurationwhich is ultimately responsible for the biological activity. In thisregard, certain amino acid substitutions are acceptable and can beexpected to be tolerated. For example, these substitutions can be inregions of the protein that are not critical to activity. Analyzing thecrystal structure of a protein, and software-based protein structuremodeling, can be used to identify regions of a protein that can bemodified (using site-directed mutagenesis, shuffling, etc.) to actuallychange the properties and/or increase the functionality of the protein.

[0181] Various properties and targeted 3D features of the protein canalso be changed without adversely affecting the toxinactivity/functionality of the protein. Conservative amino acidsubstitutions can be expected to be tolerated/to not adversely affectthe three-dimensional configuration of the molecule. Amino acids can beplaced in the following classes: non-polar, uncharged polar, basic, andacidic. Conservative substitutions whereby an amino acid of one class isreplaced with another amino acid of the same type fall within the scopeof the subject invention so long as the substitution is not adverse tothe biological activity of the compound. Table 1 provides a listing ofexamples of amino acids belonging to each class. TABLE 1 Class of AminoAcid Examples of Amino Acids Nonpolar Ala, Val, Leu, Ile, Pro, Met, Phe,Trp Uncharged Polar Gly, Ser, Thr, Cys, Tyr, Asn, Gln Acidic Asp, GluBasic Lys, Arg, His

[0182] In some instances, non-conservative substitutions can also bemade. The critical factor is that these substitutions must notsignificantly detract from the functional/biological activity of thetoxin.

[0183] As used herein, reference to “isolated” polynucleotides and/or“purified” toxins refers to these molecules when they are not associatedwith the other molecules with which they would be found in nature. Thus,reference to “isolated” and/or “purified” signifies the involvement ofthe “hand of man” as described herein. For example, a bacterial toxin“gene” of the subject invention put into a plant for expression is an“isolated polynucleotide.” Likewise, a Paenibacillus protein,exemplified herein, produced by a plant is an “isolated protein.”

[0184] Because of the degeneracy/redundancy of the genetic code, avariety of different DNA sequences can encode the amino acid sequencesdisclosed herein. It is well within the skill of a person trained in theart to create alternative DNA sequences that encode the same, oressentially the same, toxins. These variant DNA sequences are within thescope of the subject invention.

[0185] Optimization of sequence for expression in plants. To obtain highexpression of heterologous genes in plants it may be preferred toreengineer said genes so that they are more efficiently expressed in(the cytoplasm of) plant cells. Maize is one such plant where it may bepreferred to re-design the heterologous gene(s) prior to transformationto increase the expression level thereof in said plant. Therefore, anadditional step in the design of genes encoding a bacterial toxin isreengineering of a heterologous gene for optimal expression.

[0186] One reason for the reengineering of a bacterial toxin forexpression in maize is due to the non-optimal G+C content of the nativegene. For example, the very low G+C content of many native bacterialgene(s) (and consequent skewing towards high A+T content) results in thegeneration of sequences mimicking or duplicating plant gene controlsequences that are known to be highly A+T rich. The presence of someA+T-rich sequences within the DNA of gene(s) introduced into plants(e.g., TATA box regions normally found in gene promoters) may result inaberrant transcription of the gene(s). On the other hand, the presenceof other regulatory sequences residing in the transcribed mRNA (e.g.,polyadenylation signal sequences (AAUAAA), or sequences complementary tosmall nuclear RNAs involved in pre-mRNA splicing) may lead to RNAinstability. Therefore, one goal in the design of genes encoding abacterial toxin for maize expression, more preferably referred to asplant optimized gene(s), is to generate a DNA sequence having a higherG+C content, and preferably one close to that of maize genes coding formetabolic enzymes. Another goal in the design of the plant optimizedgene(s) encoding a bacterial toxin is to generate a DNA sequence inwhich the sequence modifications do not hinder translation.

[0187] The table below (Table 2) illustrates how high the G+C content isin maize. For the data in Table 2, coding regions of the genes wereextracted from GenBank (Release 71) entries, and base compositions werecalculated using the MacVector™ program (Accelerys, Burlington, Mass.).Intron sequences were ignored in the calculations.

[0188] Due to the plasticity afforded by the redundancy/degeneracy ofthe genetic code (i.e., some amino acids are specified by more than onecodon), evolution of the genomes in different organisms or classes oforganisms has resulted in differential usage of redundant codons. This“codon bias” is reflected in the mean base composition of protein codingregions. For example, organisms with relatively low G+C contents utilizecodons having A or T in the third position of redundant codons, whereasthose having higher G+C contents utilize codons having G or C in thethird position. It is thought that the presence of “minor” codons withina mRNA may reduce the absolute translation rate of that mRNA, especiallywhen the relative abundance of the charged tRNA corresponding to theminor codon is low. An extension of this is that the diminution oftranslation rate by individual minor codons would be at least additivefor multiple minor codons. Therefore, mRNAs having high relativecontents of minor codons would have correspondingly low translationrates. This rate would be reflected by subsequent low levels of theencoded protein.

[0189] In reengineering genes encoding a bacterial toxin for maize (orother plant, such as cotton or soybean) expression, the codon bias ofthe plant has been determined. The codon bias for maize is thestatistical codon distribution that the plant uses for coding itsproteins and the preferred codon usage is shown in Table 3. Afterdetermining the bias, the percent frequency of the codons in the gene(s)of interest is determined. The primary codons preferred by the plantshould be determined as well as the second and third choice of preferredcodons. Afterwards, the amino acid sequence of the bacterial toxin ofinterest is reverse translated so that the resulting nucleic acidsequence codes for exactly the same protein as the native gene wantingto be heterologously expressed. The new DNA sequence is designed usingcodon bias information so that it corresponds to the most preferredcodons of the desired plant. The new sequence is then analyzed forrestriction enzyme sites that might have been created by themodification. The identified sites are further modified by replacing thecodons with second or third choice preferred codons. Other sites in thesequence which could affect transcription or translation of the gene ofinterest are the exon:intron junctions (5′ or 3′), poly A additionsignals, or RNA polymerase termination signals. The sequence is furtheranalyzed and modified to reduce the frequency of TA or GC doublets. Inaddition to the doublets, G or C sequence blocks that have more thanabout four residues that are the same can affect transcription of thesequence. Therefore, these blocks are also modified by replacing thecodons of first or second choice, etc. with the next preferred codon ofchoice. TABLE 2 Compilation of G + C contents of protein coding regionsof maize genes Protein Class.sup.a Range % G + C Mean % G + C.sup.bMetabolic Enzymes (76) 44.4-75.3 59.0 (. + −.8.0) Structural Proteins(18) 48.6-70.5 63.6 (. + −.6.7) Regulatory Proteins (5) 57.2-68.8 62.0(. + −.4.9) Uncharacterized Proteins (9) 41.5-70.3 64.3 (. + −.7.2) AllProteins (108) 44.4-75.3 60.8 (. + −.5.2)

[0190] It is preferred that the plant optimized gene(s) encoding abacterial toxin contain about 63% of first choice codons, between about22% to about 37% second choice codons, and between about 15% to about 0%third choice codons, wherein the total percentage is 100%. Mostpreferred the plant optimized gene(s) contains about 63% of first choicecodons, at least about 22% second choice codons, about 7.5% third choicecodons, and about 7.5% fourth choice codons, wherein the totalpercentage is 100%. The preferred codon usage for engineering genes formaize expression are shown in Table 3. The method described aboveenables one skilled in the art to modify gene(s) that are foreign to aparticular plant so that the genes are optimally expressed in plants.The method is further illustrated in PCT application WO 97/13402.

[0191] In order to design plant optimized genes encoding a bacterialtoxin, the amino acid sequence of said protein is reverse translatedinto a DNA sequence utilizing a non-redundant genetic code establishedfrom a codon bias table compiled for the gene sequences for theparticular plant, as shown in Table 2. The resulting DNA sequence, whichis completely homogeneous in codon usage, is further modified toestablish a DNA sequence that, besides having a higher degree of codondiversity, also contains strategically placed restriction enzymerecognition sites, desirable base composition, and a lack of sequencesthat might interfere with transcription of the gene, or translation ofthe product mRNA. TABLE 3 Preferred amino acid codons for proteinsexpressed in maize Amino Acid Godon* Alanine GGG/GGG Gysteine TGG/TGTAspartic Acid GAC/GAT Glutamic Acid GAG/GAA Phenylalanine TTC/TTTGlycine GGC/GGG Histidine GAG/CAT Isoleucine ATC/ATT Lysine AAG/AAALeucine GTG/CTG Methionine ATG Asparagine AAG/AAT Proline GCG/CCAGlutamine GAG/CAA Arginine AGG/CGC Serine AGC/TGG Threonine AGG/AGGValine GTG/GTC Tryptophan TGG Tryrosine TAG/TAT Stop TGA/TAG

[0192] Thus, synthetic genes that are functionally equivalent to thetoxins/genes of the subject invention can be used to transform hosts,including plants. Additional guidance regarding the production ofsynthetic genes can be found in, for example, U.S. Pat. No. 5,380,831.

[0193] In some cases, especially for expression in plants, it can beadvantageous to use truncated genes that express truncated proteins.Höfte et al. 1989, for example, discussed in the Background Sectionabove, discussed protoxin and core toxin segments of B.t. toxins.Preferred truncated genes will typically encode 40, 41, 42, 43, 44, 45,46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63,64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81,82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or99% of the full-length toxin.

[0194] Transgenic hosts. The toxin-encoding genes of the subjectinvention can be introduced into a wide variety of microbial or planthosts. In preferred embodiments, transgenic plant cells and plants areused. Preferred plants (and plant cells) are corn, maize, and cotton.

[0195] In preferred embodiments, expression of the toxin gene results,directly or indirectly, in the intracellular production (andmaintenance) of the pesticide proteins. Plants can be renderedinsect-resistant in this manner. Whentransgenic/recombinant/transformed/transfected host cells (or contentsthereof) are ingested by the pests, the pests will ingest the toxin.This is the preferred manner in which to cause contact of the pest withthe toxin. The result is control (killing or making sick) of the pest.Sucking pests can also be controlled in a similar manner. Alternatively,suitable microbial hosts, e.g., Pseudomonas such as P. fluorescens, canbe applied where target pests are present; the microbes can proliferatethere, and are ingested by the target pests. The microbe hosting thetoxin gene can be treated under conditions that prolong the activity ofthe toxin and stabilize the cell. The treated cell, which retains thetoxic activity, can then be applied to the environment of the targetpest.

[0196] Where the toxin gene is introduced via a suitable vector into amicrobial host, and said host is applied to the environment in a livingstate, certain host microbes should be used. Microorganism hosts areselected which are known to occupy the “phytosphere” (phylloplane,phyllosphere, rhizosphere, and/or rhizoplane) of one or more crops ofinterest. These microorganisms are selected so as to be capable ofsuccessfully competing in the particular environment (crop and otherinsect habitats) with the wild-type microorganisms, provide for stablemaintenance and expression of the gene expressing the polypeptidepesticide, and, desirably, provide for improved protection of thepesticide from environmental degradation and inactivation.

[0197] A large number of microorganisms are known to inhabit thephylloplane (the surface of the plant leaves) and/or the rhizosphere(the soil surrounding plant roots) of a wide variety of important crops.These microorganisms include bacteria, algae, and fungi. Of particularinterest are microorganisms, such as bacteria, e.g., genera Pseudomonas,Erwinia, Serratia, Klebsiella, Xanthomonas, Streptomyces, Rhizobium,Rhodopseudomonas, Methylophilius, Agrobacterium, Acetobacter,Lactobacillus, Arthrobacter, Azotobacter, Leuconostoc, and Alcaligenes;fungi, particularly yeast, e.g., genera Saccharomyces, Cryptococcus,Kluyveromyces, Sporobolomyces, Rhodotorula, and Aureobasidium. Ofparticular interest are such phytosphere bacterial species asPseudomonas syringae, Pseudomonas fluorescens, Serratia marcescens,Acetobacter xylinum, Agrobacterium tumefaciens, Rhodopseudomonasspheroides, Xanthomonas campestris, Rhizobium melioti, Alcaligenesentrophus, and Azotobacter vinlandii; and phytosphere yeast species suchas Rhodotorula rubra, R. glutinis, R. marina, R. aurantiaca,Cryptococcus albidus, C. diffluens, C. laurentii, Saccharomyces rosei,S. pretoriensis, S. cerevisiae, Sporobolomyces roseus, S. odorus,Kluyveromyces veronae, and Aureobasidium pollulans. Also of interest arepigmented microorganisms.

[0198] Insertion of genes to form transgenic hosts. One aspect of thesubject invention is the transformation/transfection of plants, plantcells, and other host cells with polynucleotides of the subjectinvention that express proteins of the subject invention. Plantstransformed in this manner can be rendered resistant to attack by thetarget pest(s).

[0199] A wide variety of methods are available for introducing a geneencoding a pesticidal protein into the target host under conditions thatallow for stable maintenance and expression of the gene. These methodsare well known to those skilled in the art and are described, forexample, in U.S. Pat. No. 5,135,867.

[0200] For example, a large number of cloning vectors comprising areplication system in E. coli and a marker that permits selection of thetransformed cells are available for preparation for the insertion offoreign genes into higher plants. The vectors comprise, for example,pBR322, pUC series, M13mp series, pACYC184, etc. Accordingly, thesequence encoding the toxin can be inserted into the vector at asuitable restriction site. The resulting plasmid is used fortransformation into E. coli. The E. coli cells are cultivated in asuitable nutrient medium, then harvested and lysed. The plasmid isrecovered. Sequence analysis, restriction analysis, electrophoresis, andother biochemical-molecular biological methods are generally carried outas methods of analysis. After each manipulation, the DNA sequence usedcan be cleaved and joined to the next DNA sequence. Each plasmidsequence can be cloned in the same or other plasmids. Depending on themethod of inserting desired genes into the plant, other DNA sequencesmay be necessary. If, for example, the Ti or Ri plasmid is used for thetransformation of the plant cell, then at least the right border, butoften the right and the left border of the Ti or Ri plasmid T-DNA, hasto be joined as the flanking region of the genes to be inserted. The useof T-DNA for the transformation of plant cells has been intensivelyresearched and described in EP 120 516; Hoekema (1985) In: The BinaryPlant Vector System, Offset-durkkerij Kanters B. V., Alblasserdam,Chapter 5; Fraley et al., Crit. Rev. Plant Sci. 4:1-46; and An et al.(1985) EMBO J. 4:277-287.

[0201] A large number of techniques are available for inserting DNA intoa plant host cell. Those techniques include transformation with T-DNAusing Agrobacterium tumefaciens or Agrobacterium rhizogenes astransformation agent, fusion, injection, biolistics (microparticlebombardment), or electroporation as well as other possible methods. IfAgrobacteria are used for the transformation, the DNA to be inserted hasto be cloned into special plasmids, namely either into an intermediatevector or into a binary vector. The intermediate vectors can beintegrated into the Ti or Ri plasmid by homologous recombination owingto sequences that are homologous to sequences in the T-DNA. The Ti or Riplasmid also comprises the vir region necessary for the transfer of theT-DNA. Intermediate vectors cannot replicate themselves in Agrobacteria.The intermediate vector can be transferred into Agrobacteriumtumefaciens by means of a helper plasmid (conjugation). Binary vectorscan replicate themselves both in E. coli and in Agrobacteria. Theycomprise a selection marker gene and a linker or polylinker which areframed by the right and left T-DNA border regions. They can betransformed directly into Agrobacteria (Holsters et al. [1978] Mol. Gen.Genet. 163:181-187). The Agrobacterium used as host cell is to comprisea plasmid carrying a vir region. The vir region is necessary for thetransfer of the T-DNA into the plant cell. Additional T-DNA may becontained. The bacterium so transformed is used for the transformationof plant cells. Plant explants can advantageously be cultivated withAgrobacterium tumefaciens or Agrobacterium rhizogenes for the transferof the DNA into the plant cell. Whole plants can then be regeneratedfrom the infected plant material (for example, pieces of leaf, segmentsof stalk, roots, but also protoplasts or suspension-cultivated cells) ina suitable medium, which may contain antibiotics or biocides forselection. The plants so obtained can then be tested for the presence ofthe inserted DNA. No special demands are made of the plasmids in thecase of injection and electroporation. It is possible to use ordinaryplasmids, such as, for example, pUC derivatives.

[0202] The transformed cells grow inside the plants in the usual manner.They can form germ cells and transmit the transformed trait(s) toprogeny plants. Such plants can be grown in the normal manner andcrossed with plants that have the same transformed hereditary factors orother hereditary factors. The resulting hybrid individuals have thecorresponding phenotypic properties.

[0203] In some preferred embodiments of the invention, genes encodingthe bacterial toxin are expressed from transcriptional units insertedinto the plant genome. Preferably, said transcriptional units arerecombinant vectors capable of stable integration into the plant genomeand enable selection of transformed plant lines expressing mRNA encodingthe proteins.

[0204] Once the inserted DNA has been integrated in the genome, it isrelatively stable there (and does not come out again). It normallycontains a selection marker that confers on the transformed plant cellsresistance to a biocide or an antibiotic, such as kanamycin, G418,bleomycin, hygromycin, or chloramphenicol, inter alia. The individuallyemployed marker should accordingly permit the selection of transformedcells rather than cells that do not contain the inserted DNA. Thegene(s) of interest are preferably expressed either by constitutive orinducible promoters in the plant cell. Once expressed, the mRNA istranslated into proteins, thereby incorporating amino acids of interestinto protein. The genes encoding a toxin expressed in the plant cellscan be under the control of a constitutive promoter, a tissue-specificpromoter, or an inducible promoter.

[0205] Several techniques exist for introducing foreign recombinantvectors into plant cells, and for obtaining plants that stably maintainand express the introduced gene. Such techniques include theintroduction of genetic material coated onto microparticles directlyinto cells (U.S. Pat. No. 4,945,050 to Cornell and U.S. Pat. No.5,141,131 to DowElanco, now Dow AgroSciences, LLC). In addition, plantsmay be transformed using Agrobacterium technology, see U.S. Pat. No.5,177,010 to University of Toledo; U.S. Pat. No. 5,104,310 to Texas A&M;European Patent Application 0131624B1; European Patent Applications120516, 159418B1 and 176,112 to Schilperoot; U.S. Pat. Nos. 5,149,645,5,469,976, 5,464,763 and 4,940,838 and 4,693,976 to Schilperoot;European Patent Applications 116718, 290799, 320500 all to Max Planck;European Patent Applications 604662 and 627752, and U.S. Pat. No.5,591,616, to Japan Tobacco; European Patent Applications 0267159 and0292435, and U.S. Pat. No. 5,231,019, all to Ciba Geigy, now Novartis;U.S. Pat. Nos. 5,463,174 and 4,762,785, both to Calgene; and U.S. Pat.Nos. 5,004,863 and 5,159,135, both to Agracetus. Other transformationtechnology includes whiskers technology. See U.S. Pat. Nos. 5,302,523and 5,464,765, both to Zeneca. Electroporation technology has also beenused to transform plants. See WO 87/06614 to Boyce Thompson Institute;U.S. Pat. Nos. 5,472,869 and 5,384,253, both to Dekalb; and WO 92/09696and WO 93/21335, both to Plant Genetic Systems. Furthermore, viralvectors can also be used to produce transgenic plants expressing theprotein of interest. For example, monocotyledonous plant can betransformed with a viral vector using the methods described in U.S. Pat.Nos. 5,569,597 to Mycogen Plant Science and Ciba-Giegy, now Novartis, aswell as U.S. Pat. Nos. 5,589,367 and 5,316,931, both to Biosource.

[0206] As mentioned previously, the manner in which the DNA construct isintroduced into the plant host is not critical to this invention. Anymethod which provides for efficient transformation may be employed. Forexample, various methods for plant cell transformation are describedherein and include the use of Ti or Ri-plasmids and the like to performAgrobacterium mediated transformation. In many instances, it will bedesirable to have the construct used for transformation bordered on oneor both sides by T-DNA borders, more specifically the right border. Thisis particularly useful when the construct uses Agrobacterium tumefaciensor Agrobacterium rhizogenes as a mode for transformation, although T-DNAborders may find use with other modes of transformation. WhereAgrobacterium is used for plant cell transformation, a vector may beused which may be introduced into the host for homologous recombinationwith T-DNA or the Ti or Ri plasmid present in the host. Introduction ofthe vector may be performed via electroporation, tri-parental mating andother techniques for transforming gram-negative bacteria which are knownto those skilled in the art. The manner of vector transformation intothe Agrobacterium host is not critical to this invention. The Ti or Riplasmid containing the T-DNA for recombination may be capable orincapable of causing gall formation, and is not critical to saidinvention so long as the vir genes are present in said host.

[0207] In some cases where Agrobacterium is used for transformation, theexpression construct being within the T-DNA borders will be insertedinto a broad spectrum vector such as pRK2 or derivatives thereof asdescribed in Ditta et al., (PNAS USA (1980) 77:7347-7351 and EPO 0 120515, which are incorporated herein by reference. Included within theexpression construct and the T-DNA will be one or more markers asdescribed herein which allow for selection of transformed Agrobacteriumand transformed plant cells. The particular marker employed is notessential to this invention, with the preferred marker depending on thehost and construction used.

[0208] For transformation of plant cells using Agrobacterium, explantsmay be combined and incubated with the transformed Agrobacterium forsufficient time to allow transformation thereof. After transformation,the Agrobacteria are killed by selection with the appropriate antibioticand plant cells are cultured with the appropriate selective medium. Oncecalli are formed, shoot formation can be encouraged by employing theappropriate plant hormones according to methods well known in the art ofplant tissue culturing and plant regeneration. However, a callusintermediate stage is not always necessary. After shoot formation, saidplant cells can be transferred to medium which encourages root formationthereby completing plant regeneration. The plants may then be grown toseed and said seed can be used to establish future generations.Regardless of transformation technique, the gene encoding a bacterialtoxin is preferably incorporated into a gene transfer vector adapted toexpress said gene in a plant cell by including in the vector a plantpromoter regulatory element, as well as 3′ non-translatedtranscriptional termination regions such as Nos and the like.

[0209] In addition to numerous technologies for transforming plants, thetype of tissue which is contacted with the foreign genes may vary aswell. Such tissue would include but would not be limited to embryogenictissue, callus tissue types I, II, and III, hypocotyl, meristem, roottissue, tissues for expression in phloem, and the like. Almost all planttissues may be transformed during dedifferentiation using appropriatetechniques described herein.

[0210] As mentioned above, a variety of selectable markers can be used,if desired. Preference for a particular marker is at the discretion ofthe artisan, but any of the following selectable markers may be usedalong with any other gene not listed herein which could function as aselectable marker. Such selectable markers include but are not limitedto aminoglycoside phosphotransferase gene of transposon Tn5 (Aph II)which encodes resistance to the antibiotics kanamycin, neomycin andG418, as well as those genes which encode for resistance or tolerance toglyphosate; hygromycin; methotrexate; phosphinothricin (bialaphos);imidazolinones, sulfonylureas and triazolopyrimidine herbicides, such aschlorsulfuron; bromoxynil, dalapon and the like.

[0211] In addition to a selectable marker, it may be desirous to use areporter gene. In some instances a reporter gene may be used with orwithout a selectable marker. Reporter genes are genes that are typicallynot present in the recipient organism or tissue and typically encode forproteins resulting in some phenotypic change or enzymatic property.Examples of such genes are provided in K. Wising et al. Ann. Rev.Genetics, 22, 421 (1988). Preferred reporter genes include thebeta-glucuronidase (GUS) of the uidA locus of E. coli, thechloramphenicol acetyl transferase gene from Tn9 of E. coli, the greenfluorescent protein from the bioluminescent jellyfish Aequorea victoria,and the luciferase genes from firefly Photinus pyralis. An assay fordetecting reporter gene expression may then be performed at a suitabletime after said gene has been introduced into recipient cells. Apreferred such assay entails the use of the gene encodingbeta-glucuronidase (GUS) of the uidA locus of E. coli as described byJefferson et al., (1987 Biochem. Soc. Trans. 15, 17-19) to identifytransformed cells.

[0212] In addition to plant promoter regulatory elements, promoterregulatory elements from a variety of sources can be used efficiently inplant cells to express foreign genes. For example, promoter regulatoryelements of bacterial origin, such as the octopine synthase promoter,the nopaline synthase promoter, the mannopine synthase promoter;promoters of viral origin, such as the cauliflower mosaic virus (35S and19S), 35T (which is a re-engineered 35S promoter, see U.S. Pat. No.6,166,302, especially Example 7E) and the like may be used. Plantpromoter regulatory elements include but are not limited toribulose-1,6-bisphosphate (RUBP) carboxylase small subunit (ssu),beta-conglycinin promoter, beta-phaseolin promoter, ADH promoter,heat-shock promoters, and tissue specific promoters. Other elements suchas matrix attachment regions, scaffold attachment regions, introns,enhancers, polyadenylation sequences and the like may be present andthus may improve the transcription efficiency or DNA integration. Suchelements may or may not be necessary for DNA function, although they canprovide better expression or functioning of the DNA by affectingtranscription, mRNA stability, and the like. Such elements may beincluded in the DNA as desired to obtain optimal performance of thetransformed DNA in the plant. Typical elements include but are notlimited to Adh-intron 1, Adh-intron 6, the alfalfa mosaic virus coatprotein leader sequence, the maize streak virus coat protein leadersequence, as well as others available to a skilled artisan. Constitutivepromoter regulatory elements may also be used thereby directingcontinuous gene expression in all cells types and at all times (e.g.,actin, ubiquitin, CaMV 35S, and the like). Tissue specific promoterregulatory elements are responsible for gene expression in specific cellor tissue types, such as the leaves or seeds (e.g., zein, oleosin,napin, ACP, globulin and the like) and these may also be used.

[0213] Promoter regulatory elements may also be active during a certainstage of the plant's development as well as active in plant tissues andorgans. Examples of such include but are not limited to pollen-specific,embryo-specific, corn-silk-specific, cotton-fiber-specific,root-specific, seed-endosperm-specific promoter regulatory elements andthe like. Under certain circumstances it may be desirable to use aninducible promoter regulatory element, which is responsible forexpression of genes in response to a specific signal, such as: physicalstimulus (heat shock genes), light (RUBP carboxylase), hormone (Em),metabolites, chemical, and stress. Other desirable transcription andtranslation elements that function in plants may be used. Numerousplant-specific gene transfer vectors are known in the art.

[0214] Standard molecular biology techniques may be used to clone andsequence the toxins described herein. Additional information may befound in Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989),Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, whichis incorporated herein by reference.

[0215] Resistance Management. With increasing commercial use ofinsecticidal proteins in transgenic plants, one consideration isresistance management. That is, there are numerous companies usingBacillus thuringiensis toxins in their products, and there is concernabout insects developing resistance to B.t. toxins. One strategy forinsect resistance management would be to combine the TC toxins producedby Xenorhabdus, Photorhabdus, and the like with toxins such as B.t.,crystal toxins, soluble insecticidal proteins from Bacillus stains (see,e.g., WO 98/18932 and WO 99/57282), or other insect toxins. Thecombinations could be formulated for a sprayable application or could bemolecular combinations. Plants could be transformed with bacterial genesthat produce two or more different insect toxins (see, e.g., Gould, 38Bioscience 26-33 (1988) and U.S. Pat. No. 5,500,365; likewise, EuropeanPatent Application 0 400 246 A1 and U.S. Pat. Nos. 5,866,784; 5,908,970;and 6,172,281 also describe transformation of a plant with two B.t.crystal toxins). Another method of producing a transgenic plant thatcontains more than one insect resistant gene would be to first producetwo plants, with each plant containing an insect resistance gene. Theseplants could then be crossed using traditional plant breeding techniquesto produce a plant containing more than one insect resistance gene.Thus, it should be apparent that the phrase “comprising apolynucleotide” as used herein means at least one polynucleotide (andpossibly more, contiguous or not) unless specifically indicatedotherwise.

[0216] Formulations and Other Delivery Systems. Formulated bait granulescontaining spores and/or crystals of the subject Paenibacillus isolate,or recombinant microbes comprising the genes obtainable from the isolatedisclosed herein, can be applied to the soil. Formulated product canalso be applied as a seed-coating or root treatment or total planttreatment at later stages of the crop cycle. Plant and soil treatmentsof cells may be employed as wettable powders, granules or dusts, bymixing with various inert materials, such as inorganic minerals(phyllosilicates, carbonates, sulfates, phosphates, and the like) orbotanical materials (powdered corncobs, rice hulls, walnut shells, andthe like). The formulations may include spreader-sticker adjuvants,stabilizing agents, other pesticidal additives, or surfactants. Liquidformulations maybe aqueous-based or non-aqueous and employed as foams,gels, suspensions, emulsifiable concentrates, or the like. Theingredients may include rheological agents, surfactants, emulsifiers,dispersants, or polymers.

[0217] As would be appreciated by a person skilled in the art, thepesticidal concentration will vary widely depending upon the nature ofthe particular formulation, particularly whether it is a concentrate orto be used directly. The pesticide will be present in at least 1% byweight and may be 100% by weight. The dry formulations will have fromabout 1-95% by weight of the pesticide while the liquid formulationswill generally be from about 1-60% by weight of the solids in the liquidphase. The formulations will generally have from about 10² to about 10⁴cells/mg. These formulations will be administered at about 50 mg (liquidor dry) to 1 kg or more per hectare.

[0218] The formulations can be applied to the environment of the pest,e.g., soil and foliage, by spraying, dusting, sprinkling, or the like.

[0219] Another delivery scheme is the incorporation of the geneticmaterial of toxins into a baculovirus vector. Baculoviruses infectparticular insect hosts, including those desirably targeted with thetoxins. Infectious baculovirus harboring an expression construct for thetoxins could be introduced into areas of insect infestation to therebyintoxicate or poison infected insects.

[0220] Insect viruses, or baculoviruses, are known to infect andadversely affect certain insects. The affect of the viruses on insectsis slow, and viruses do not immediately stop the feeding of insects.Thus, viruses are not viewed as being optimal as insect pest controlagents. However, combining the toxin genes into a baculovirus vectorcould provide an efficient way of transmitting the toxins. In addition,since different baculoviruses are specific to different insects, it maybe possible to use a particular toxin to selectively target particularlydamaging insect pests. A particularly useful vector for the toxins genesis the nuclear polyhedrosis virus. Transfer vectors using this virushave been described and are now the vectors of choice for transferringforeign genes into insects. The virus-toxin gene recombinant may beconstructed in an orally transmissible form. Baculoviruses normallyinfect insect victims through the mid-gut intestinal mucosa. The toxingene inserted behind a strong viral coat protein promoter would beexpressed and should rapidly kill the infected insect.

[0221] In addition to an insect virus or baculovirus or transgenic plantdelivery system for the protein toxins of the present invention, theproteins may be encapsulated using Bacillus thuringiensis encapsulationtechnology such as but not limited to U.S. Pat. Nos. 4,695,455;4,695,462; 4,861,595 which are all incorporated herein by reference.Another delivery system for the protein toxins of the present inventionis formulation of the protein into a bait matrix, which could then beused in above and below ground insect bait stations. Examples of suchtechnology include but are not limited to PCT Patent Application WO93/23998, which is incorporated herein by reference.

[0222] Plant RNA viral based systems can also be used to expressbacterial toxin. In so doing, the gene encoding a toxin can be insertedinto the coat promoter region of a suitable plant virus which willinfect the host plant of interest. The toxin can then be expressed thusproviding protection of the plant from insect damage. Plant RNA viralbased systems are described in U.S. Pat. Nos. 5,500,360 to Mycogen PlantSciences, Inc. and U.S. Pat. Nos. 5,316,931 and 5,589,367 to BiosourceGenetics Corp.

[0223] In addition to producing a transformed plant, there are otherdelivery systems where it may be desirable to reengineer the bacterialgene(s). For example, a protein toxin can be constructed by fusingtogether a molecule attractive to insects as a food source with a toxin.After purification in the laboratory such a toxic agent with “built-in”bait could be packaged inside standard insect trap housings.

[0224] Mutants. Mutants of the DAS1529 and DB482 isolates of theinvention can be made by procedures that are well known in the art. Forexample, an asporogenous mutant can be obtained through ethylmethanesulfonate (EMS) mutagenesis of an isolate. The mutants can be made usingultraviolet light and nitrosoguanidine by procedures well known in theart.

[0225] All patents, patent applications, provisional applications, andpublications referred to or cited herein are incorporated by referencein their entirety to the extent they are not inconsistent with theexplicit teachings of this specification.

[0226] Following are examples that illustrate procedures for practicingthe invention. These examples should not be construed as limiting. Allpercentages are by weight and all solvent mixture proportions are byvolume unless otherwise noted.

EXAMPLE 1 Isolation and Discovery of Insecticidal Activity of DAS1529 asa Paenibacillus sp.

[0227] A bacterial strain, designated DAS1529, was found to producefactors that were growth inhibitory to neonates of lepidopteran insects,corn earworm (Heliothis zea, CEW), tobacco budworm (Heliothis virescens;TBW), and tobacco hornworm (Manduca sexta; THW).

[0228] DAS1529 was cultured in 2% Protease Peptone No.3 (PP3) medium(Difco Laboratories, Detroit, Mich.) supplemented with 1.25% NaCl or inJB medium supplemented with 0.2% glucose. Bacterial culture was grown at25° C. for ˜40 hours at 150 rpm.

[0229] The insecticidally active factors were initially found in thefermentation broth that was concentrated on 5 kDa molecular weightcutoff filters. Those factors were heat labile (inactivated by heatingat 85° C. for 20 minutes). These data indicated that the factors wereproteinaceous. See also end of Example 4.

[0230] To identify active factors in cell pellets, the bacterial culturewas centrifuged at 8000 rpm at 4° C. for 15 minutes, washed once withsterile distilled water, and resuspended to 33× of the original culturevolume in sterile distilled water, and subjected to insect bioassay asdescribed below in Example 3. The bioassay data for DAS1529 strain issummarized in Table 4. The data showed that the culture broth andconcentrated DAS1529 bacterial cells conferred good activity against CEW(30 to 50% mortality at 33×) and TBW (100% mortality at 33×). Thosetoxin factors in DAS1529 have significant relevance to the developmentof commercial transgenic products targeting lepidopteran insects (e.g.CEW and TBW) in corn and cotton. TABLE 4 Bioassay of DAS 1529 StrainInsects TBW CEW Grubs Broth Activity +++ +++ n.d. Pellet Activity +++ ++−

EXAMPLE 2 Classification of DAS1529

[0231] Molecular phylogeny was performed to determine the taxonomicaffiliation of strain DAS1529. The nucleotide sequence of the 16S rDNAof DAS 1529 was determined and used for similarity and phylogeneticanalyses (using the MicroSeq Kit from ABI). The sequence is provided asSEQ ID NO:16. BLAST search results are as follows: Core value (bits) egi|15395282|emb|AJ320490.1|PTH320490 Paenibacillus thiamino. . . 29060.0 gi|3328014|gb|AF071859.1|AF071859 Paenibacillus popilliae s. . .2815 0.0 gi|3328015|gb|AF071860.1|AF071860 Paenibacillus popilliae s. .. 2815 0.0 gi|2769591|emb|Y16129.1|PS16SC168 Paenibacillus sp. C-168 1.. . 2699 0.0 gi|2769591|emb|Y16129.1|PS16SC168 Paenibacillus sp.T-168 1. . . 2509 0.0 gi|2077917|dbj|D78475.1|D78475 Paenibacillusthiaminolyticu. . . 2503 0.0 gi|3328016|gb|AF071861.1|AF071861Paenibacillus lentimorbus. . . 2493 0.0 gi|2895560|gb|AF039408.1|Bacillus tipchiralis 16S ribosoma. . . 2493 0.0gi|2077936|dbj|D88513.1|D88513 Paenibacillus thiaminolyticu. . . 24930.0 gi|15395283|emb|AJ320491.1|PAL320491 Paenibacillus alvei pa. . .2404 0.0

[0232] These same top scoring sequences from the BLAST search were alsocompared using the Gap routine (Needleman and Wunsch, J. Mol. Biol. 48;443-453 (1970)) from GCG version 10.2, with the following results: %Sim%Ident gi|15395282|emb|AJ320490.1|PTH320490 Paenibacillus thiamino. . .99.2 99.6 gi|3328014|gb|AF071859.1|AF071859 Paenibacillus popilliae s. .. 99.2 99.6 gi|3328015|gb|AF071859.1|AF071860 Paenibacillus popilliae s.. . 99.2 99.3 gi|2769591|emb|Y16129.1|PS16SC168 Paenibacillus sp.C-168 1. . . 97.1 97.3 gi|2769590|emb|Y16129.1|PS16ST168 Paenibacillussp. T-168 1. . . 97.4 97.4 gi|2077917|dbj|D78475.1|D78475 Paenibacillusthiaminolyticu. . . 96.5 98.1 gi|3328016|gb|AG071861.1|AF071861Paenibacillus lentimorbus. . . 98.8 98.9 gi|2895560|gb|AF039408.1|Bacillus tipchiralis 16S ribosoma. . . 96.0 96.9gi|2077936|dbj|D88513.1|D88513 Paenibacillus thiaminolyticu. . . 96.798.7 gi|15395283|emb|AJ320491.1|PAL320491 Paenibacillus alvei pa. . .95.2 95.3

[0233] A number of related sequences, including the top scoringsequences noted above, were also trimmed and aligned as described byShida et al. (Int. J. Syst. Bacteriol. 47:289-298, 1997), using thesequence alignment program CLUSTAL W (Thompson, J. D., D. G. Higgins,and T. J. Gibson, Nucleic Acids Res. 22:4673-4680, 1994). The resultsclearly place DAS1529 in the Paenibacillus popilliae/Paenibacilluslentimorbus subcluster of the genus Paenibacillus identified byPettersson et al. (Int. J. Syst. Bacteriol. 49:531-540, 1999), and areconsistent with the analyses reported above. This subcluster includesthe insect-associated species P. popilliae and P. lentimorbus, as wellas P. thiaminolyticus, Paenibacillus sp. T-168 and C-168, and “Bacillustipchiralis,” which are not known to have an insect association(Pettersson et al., 1999). As noted by Wayne et al. (Int. J. Syst.Bacteriol. 37:463-464, 1987) and Vandamme et al. (Microbiol. Rev.60:407-438), rDNA sequences that are greater than 97% identical cannotgenerally be used to assign a bacterial strain to a particular speciesin the absence of additional information. In the case of DAS1529,insecticidal activity on lepidoptera and evidence of a thiaminase arenot consistent with known P. popilliae and P. lentimorbus, and theinsect association is not consistent with known P. thiaminolyticus (aswell as the other subcluster species).

[0234] As other Paenibacillus strains are known causative agents ofmilky disease in larvae of Japanese beetles (Popillia jalonica; Harrisonet al., 2000), the DAS1529 was tested for activity on June beetles, arelative of Japanese beetles. No activity was found for cultures grownin JB and PP3 medium. Microscopic examination of those cultures revealedeven-colored rods with no visible sporulation or parasporal crystalspresent. We are able to show DAS1529 can sporulate in defined medium andculture conditions and within the hemolymph of Manducca sexta. It isknown that the Japanese beetle active Paenibacillus strains aretypically associated with paraspore and parasporal bodies (Harrison etal., 2000).

[0235] Additional work will be needed to determine whether DAS1529belongs to an existing species or should be assigned a new speciesdesignation.

EXAMPLE 3 Insect Bioassay Methodology

[0236] Two insect bioassay methods were used to obtain results presentedbelow. A 96-well format and a 128-well format were used for primaryscreening for activity against lepdidopteran insects. A 24-well dietincorporation format was used to determine specific activity (LC50s) ofthe toxin.

[0237] For the 96-well format, artificial diet was dispensed into96-well microtiter plates. Each well measured approximately 0.32 cm² andcontained 150 μl artificial diet. Samples/toxins were applied at a rateof 50 μl /well for fermentation broth, cell pellets, and purifiedtoxins. Positive control (Cry1Ac) at appropriate doses and negativecontrols (water, medium blank, bacterial host strains not expressingtarget toxin) at top dose were included. Samples were allowed to dry forapproximately 1-3 hours so that the samples lost their moisture but thediet retained its moisture. Either insect eggs were dispersed onto thesurface of the sample treated diet, or a single insect larva was seededper well. The infested plate was sealed either with iron-on mylarcovering or covered with sticky lidding with perforations. Tiny airholes were made in the mylar covering to ensure air supply to theinsects. The plates were incubated at 28° C. for 5 days and scored formortality and stunting. This was done on a per-well basis, ignoring thenumber of larvae per well, as multiple eggs are often deposited perwell. Activity scores were then assigned to each treatment: 0=noactivity, larvae healthy like water control wells, 1=larvae werestunted, or stunted with some mortality, 2 =larvae were all dead.

[0238] The specific activities (LC50s) of samples/toxins were determinedby diet incorporation bioassay in 24-well Nutrend trays (Nu-Trend™Container Corp., Jacksonville, Fla.). Insect artificial diet was madejust prior to use and held in liquid state at 55° C. in a water bath.Serial dilutions (≧5) were made by mixing 27 ml of artificial diet withno more than 3 ml of samples/toxins. A total of 30 ml sample and dietmixture was vortexed for 30 seconds and then evenly distributed intoeach tray, filling ˜50% of the well volume. Trays were allowed to coolfor at least 30 minutes prior to infesting. One test insect was infestedinto each well, and clear mylar was sealed over the top of each tray tocontain the insects. Small holes were punched with an insect pin in themylar over each well for air circulation. Assays were generally held at25° C. for 6 days but some may have been held at 30° C. for 4 days ifquicker results were needed. A set of positive and negative controls wasrun for each assay. Assays were graded on the basis of mortality butdata on stunting was also recorded. Statistical methods were used toestimate LC50s for assayed samples and was expressed as ng or μg/mldiet.

EXAMPLE 4 Biochemical Purification and Characterization of InsecticidalToxins from DAS1529 Fermentation Broth—Thiaminase

[0239] The fermentation broths of DAS1529 contained insecticidalactivity against lepidopteran species, such as tobacco budworm, cornearworm, and tobacco hornworm. The nature of the insecticidal activitywas investigated by biochemical purification and characterization. Cornearworm bioassay, as described in Example 3, was used during thepurification process to follow insecticidal activities.

[0240] Fermentation broths of DAS1529 were produced using 2% PP3supplemented with 1.25% NaCl and processed as described in Example 1.Four liters of broth was concentrated using an Amicon (Beverly, Mass.)spiral ultrafiltration cartridge Type S1Y10 (molecular weight cut off 10kDa) attached to an Amicon M-12 filtration device according to themanufacturer's recommendations. The retentate was diafiltered with 20 mMsodium phosphate, pH 7.0 (Buffer A) and applied at 5 ml/min to a Qcepharose XL anion exchange column (1.6×10 cm). The column was washedwith 5 bed volumes of Buffer A to remove unbound proteins. Toxinactivity was eluted by 1.0 M NaCl in Buffer A.

[0241] The fraction containing the insecticidal activity was loaded in20 ml aliquots onto a gel filtration column Macro-Prep SE 1000/40(2.6×100 cm) which was equilibrated with Buffer A.

[0242] The protein was eluted in Buffer A at a flow rate of 3 ml/min.Fractions with activity against corn earworm were pooled and wereapplied to a MonoQ (1.0×10 cm) column equilibrated with 20 mM Tris-HCl,pH 7.0 (Buffer B) at a flow rate of 1 ml/min. The proteins bound to thecolumn were eluted with a linear gradient of 0 to 1 M NaCl in Buffer Bat 2 ml/min for 60 min. Two milliliter fractions were collected andactivity was determined as described in Example 1.

[0243] Solid (NH₄)₂SO₄ was added to the above active protein fractionsto a final concentration of 1.7 M. Proteins were then applied to aphenyl-Superose (1.0×10 cm) column equilibrated with 1.7 M (NH₄)₂SO₄ in50 mM potassium phosphate buffer, pH 7 (Buffer C) at 1 ml/min. Afterwashing the column with 10 milliliters of Buffer C, proteins bound tothe column were eluted with a linear gradient Buffer C to 5 mM potassiumphosphate, pH 7.0 at 1 ml/min for 120 min. The most active fractionsdetermined by bioassay were pooled and dialyzed overnight against BufferA.

[0244] The dialyzed sample was applied to a Mono Q (0.5×5 cm) columnwhich was equilibrated with Buffer B at 1 ml/min. The proteins bound tothe column were eluted at 1 ml/min by a linear gradient of 0 to 1 M NaClin Buffer B. The active fractions were pooled and adjusted to a final(NH₄)₂SO₄ concentration of 1.7M. Proteins were then applied to aphenyl-Superose (0.5.0×5 cm) column equilibrated with Buffer C at 1ml/min. Proteins bound to the column were eluted with a linear gradientof Buffer C to 5 mM potassium phosphate, pH 7.0 at 0.5 ml/min for 40min. The purified fractions were pooled and dialyzed overnight againstBuffer A.

[0245] The molecular weight of the final purified toxin was examined bya gel-filtration column Superdex S-200. The toxin exhibited a nativemolecular weight of approximately 40 kDa. SDS-PAGE of the purifiedtoxins showed a predominant band of approximately 40 kDa. This suggestedthat the native DAS1529 toxin (in this fraction) was an approximately 40kDa monomer.

[0246] The purified toxin was electrophoresed in 4-20% SDS-PAGE andtransblotted to PVDF membrane. The blot underwent amino acid analysisand N-terminal amino acid sequencing (SEQ ID NO.17). Searching proteindatabase (NCBI-NR) using the sequence as a query revealed that it is 95%identical to the approximately 42 kDa thiaminase I from Bacillusthiaminolyticus (Campobasso et al., 1998; GENBANK Accession No. 2THIA;SEQ ID NO:18). Partial sequence alignments are illustrated in FIG. 3,which would be the same alignment with GENBANK Accession No. AAC44156(thiaminase I precursor; U17168 is the corresponding entry in GENBANKfor the DNA, which could be expressed to get a thiaminase produced andsecreted from a bacterial cell). The purified thiaminase from DAS1529was tested on corn earworm (CEW), the results were shown in FIG. 4. Thistoxin did not kill corn earworm (up to a concentration of 8 μg/cm²) butexhibited 95% growth inhibition at a concentration as low as 5 ng/cm².It was also found that the purified thiaminase was not deactivated byproteinase K.

EXAMPLE 5 Cloning of Genes Encoding Insecticidal Factors Produced by DAS1529

[0247] In an attempt to clone the nucleotide sequence(s) that encode theinsecticidal factor(s) produced by DAS 1529, a cosmid library wasconstructed using total DNA isolated from DAS 1529 and was screened forinsecticidal activity. Six recombinant cosmid clones were identifiedthat produced insecticidal activity against corn earworm and tobaccobudworm neonates. Three of the cosmid clones produced heat labile (whenheated at 85° C. for 20 minutes) factors that resulted in insectmortality. The other three cosmid clones produced heat labile factorsthat were growth inhibitory to insects. One of the cosmids that producedinsect mortality, designated as cosmid SB12, was chosen for nucleotidesequence analysis.

[0248] A. Construction of a Cosmid Library of DAS1529.

[0249] Total DNA was isolated from DAS1529 with a DNA purification kit(Qiagen Inc., Valencia, Calif.). Vector and insert DNA preparation,ligation, and packaging, followed instructions from the supplier(Stratagene, La Jolla, Calif.). The DAS 1529 DNA inserts as Sau3AI DNAfragments were cloned into the BamHI site of SuperCos 1 cosmid vector.The ligated product was packaged with the Gigapack® III gold packagingextract and transfected into host cells XL1-Blue MRF′. Transformantswere selected on LB-kanamycin agar plates. The cosmid library consistedof 960 randomly picked colonies that were grown in ten 96-wellmicrotiter plates in 200 μl LB-kanamycin (50 μg/ml) for insect activityscreening and long term storage.

[0250] B. Screening of DAS1529 Cosmid Library for Insecticidal Activity

[0251] For the primary screening for clones active against lepdidopteraninsects (CEW and TBW), a total of 960 cosmid clones as single colonieswere grown in 2 ml cultures in 96 well plates. Cultures were spun andre-suspended in original culture media at approximately 10×concentration and submitted to bioassay. The SuperCos 1 vector (SB1) wasincluded as a negative control. Sixteen positive clones (SB2 to SB17)were isolated from the first round of screening. Second and third roundsof screening were carried out to screen for activity against TBW andCEW; one cosmid clone (SB12) consistently showing the highest activitywas chosen for further analysis. Table 5 summarizes the activityspectrum (as tested) of the SB12 cosmid. (BAW is beet armyworm,Spodoptera exigua; ECB is European comborer, Ostrinia nubilalis; SCRW isSouthern corn rootworm, Diabrotica undecimpucata howardi.) The broth ofSB12 E. coli culture both contained no CEW activity; hence, the activefactors in SB12 were different from the active factors in DAS1529 strainculture broth. TABLE 5 Bioassay of SB12 E. coli Clone Insects TBW CEWECB BAW Grubs SCR Broth Activity − − n.d. n.d. n.d. n.d. Pellet Activity+++ ++ + ++ − −

[0252] C. Sequencing of SB12 Cosmid Insert and Identification of tc- andcry-like ORFs.

[0253] Nucleotide sequencing of cosmid SB12 showed that it contained agenomic insert of approximately 34 kb. Analysis of this sequencesurprisingly revealed the presence of at least 10 putative open readingframes (ORFs) (see FIG. 2). Six of the identified ORFs were surprisinglyfound to have some degree of amino acid sequence identity (38-48%) totcaA, tcaB, tcaC, and tccC previously identified from Photorhabdusluminescens (Waterfield et al., 2001), Xenorhabdus nematophilus (Morganet al., 2001), Serratia entomophila (Hurst and Glare, 2002; Hurst etal., 2000), and Yersinia pestis (Cronin et al., 2001). Those TC proteingenes from Photorhabdus, Xenorhabdus, and Serratia have been shown toencode insecticidal factors. Also very interesting was that one DAS 1529ORF had ˜40% amino acid sequence identity to Cry1Ac from Bacillusthuringiensis, another gene previously identified as an insecticidalfactor (Schnepf et al., 1998; de Maagd et al., 2001). Those findingshave significant implication in understanding toxin gene distribution inthe bacterial kingdom and in developing further strategies for toxingene mining and engineering.

[0254] The nucleotide sequence of the SB12 cosmid was determined. Theassembled DNA of 41,456 bp long was further analyzed. Three gaps werelocated: two in the cosmid vector and one in the insert. Analysis of thenucleotide sequence of the longest contig of approximately 34,000 bprevealed the presence of at least 10 putative open reading frames(ORFs), identified as potential start codons followed by extended openreading frames. This method is known to misidentify translational startsites by 19% (Bacillus subtilis) and 22% (Bacillus halodurans) ingenomes related to Paenibacillus (Besemer, J., Lomsadze, A., Borodovsky,M., Nucleic Acids Res. 29:2607-2618, 2001). Therefore, the quality andposition of bases complementary to the B. subtilis 16S rRNA 5′ end(reviewed in Rocha, E. P. C., Danchin, A., Viari, A., Nucleic Acids Res.27:3567-3576, 1999), N-terminal amino acid sequencing, and alignments torelated genes were considered in identifying the native translationinitiation sites. The putative ORFs and annotations are summarized inTable 6 and are discussed in more detail below. TABLE 6 Sequenceannotation for SB12 cosmid sequence Sequence SEQ Some ORF ORF LocationID similarity Designation on SEQ NO: to: on SB12 Comments ID NO: 1 1Entire   (1-33521) insert of SB12 2 tcaA ORF1   1-3264 (truncated at 5′)3 Translation   (1-3261) of ORF 1 4 tcaB ORF2 3271-4740 (with IS (5′end); element 6079-8226 removed) (3′ end) 5 Translation (amino acids ofORF2   1-490/ (without   491-1205) insertion) from 5′- most ATG 6 tcaAORF3  9521-12820 7 Translation  (9521-12817) of ORF3 8 tcaB ORF412827-16453 9 Translation (12827-16450) of ORF4 from 5′- most ATG 10tcaC ORF5 16516-20850 11 Translation (16516-20847) of ORF5 12 tccC ORF620867-23659 13 Translation (20867-23656) of ORF6 (from better RBS) 14ORF7 24422-26213 (Cry1529) 15 Translation of ORF7 19 tccC Translation20798-23656 from 5′- most ATG of ORF6

[0255] ORF1 begins with the first nucleotide of the cloning site for theDAS1529 DNA in cosmid SB12, and is therefore missing its nativetranslation initiation site. ORF1 shares significant DNA sequencehomology with ORF3, and sequence comparison analysis suggests the first18 bp of ORF1 is truncated, and that the first six codons encode theamino acids Met-Val-Ser-Thr-Thr, as found in OFR3. The ORF1 translationstart is presumably similar to that of ORF3, from approximately bases9505 through 9523 of SEQ ID NO:1. Two predicted amino acid sequences arepresented for ORF2, ORF4, and ORF6 (SEQ ID NOs:19 and 13), based onalternative translation initiation sites, as noted above. For ORF2, SEQID NO:5 is discussed above. The alternate, and preferred, start site isat residue 3295 of ORF1. Thus, the protein resulting from this startsite would begin at amino acid residue 9 of SEQ ID NO:5 (translationfrom better RBS). Likewise, for ORF4, SEQ ID NO:9 is discussed above.The alternate, and preferred, start site is at residue 12,852 of SEQ IDNO:1. The resulting protein would also be missing the first eight aminoacids of SEQ ID NO:9 (thus beginning with amino acid residue 9 of SEQ IDNO:8—translation from better RBS).

EXAMPLE 6 Sequence Analysis of “Duplicated” TCs

[0256] The degree of sequence identity for the two ORF2 fragments(tcaB₁) compared to ORF4 (tcaB₂) was determined, as was that for ORF1(tcaA₁) compared to ORF3 (tcaA₂). A similar sequence relationship wasobserved for both pairs of ORFs.

[0257] ORF2 was constructed by combining two fragments, because of aninsertion sequence-like element which was inserted in nature (apparentlyspontaneously), and disrupted this ORF. See FIG. 2. The location of thisinsertion is determinable by analyzing/comparing the entire SB12 DNAsequence (SEQ ID NO:1) with the sequence of SEQ ID NO:4, the latter ofwhich does not reflect the (non-coding) insertion. As indicated withbrackets in FIG. 7, the sequence of the 5′ translation product prior toresidue 490 of SEQ ID NO:4 and the 3′ translation product from residue491 on, align well with ORF4 (SEQ ID NO:8). The DNA sequence at theapparent insertion point shows a 9 bp direct repeat commonly foundflanking insertion elements (CACCGAGCA, bases 4734-4742 and 6071-6080 ofSEQ ID NO:1).

EXAMPLE 7 Further Sequence Analysis

[0258] In summary, according to Vector NTI clustalW, GCG, and/or Blastpanalyses, six of the identified ORFs (ORF1 to ORF6) had 38-48% aminoacids sequence identity to tcaA, tcaB, tcaC, and tccC (previouslyidentified Photorhabdus tc genes). The ORF7 encoded a protein thatshared ˜40% amino acid sequence identity to Cry1Ac from Bacillusthuringiensis, another gene previously identified as an insecticidalfactor. A phylogram was generated by incorporating ORF7 (Cry1529)sequence with a large number of other Cry proteins (FIG. 8). Thisphylogenetic tree suggests that Cry1529 is distantly related to other P.popilliae Cry sequences such as the Cry18s (Zhang et al., 1997, Zhang etal., 1998) that are closer to Cry2s; Cry1529 falls (remotely but mostclosely) into a group of Cry proteins containing commonly foundlepidoptera (Cry1, Cry9), coleoptera (Cry3, Cry8, Cry7), and diptera(Cry4) toxins, which is a distinct group compared to those includingnematode toxins Cry5, -12, -13, -14, and -21 and Cry2, -18.

[0259] It was a surprising and novel discovery to find Cry and TCprotein genes (in the SB12 genomic insert) in Paenibacillus. Theidentification of new Cry and TC protein genes has relevance to theart's understanding of Photorhabdus and Xenorhabdus, and Bacillusthuringiensis, and broadens the scope of bacterial genera in which Cryand TC protein genes have been found. The size of the full-lengthCry1529 identified herein corresponds to the core toxin of Cry1s;Cry1529 represents a new class of Cry proteins which also hasimplications for isolating further cry genes from Bacillus thuringiensisand Paenibacillus.

[0260] To verify that these surprising observations were not the resultof strain contamination (i.e., to confirm that the 34 kb insert carryingTC and Cry ORFs was indeed from the total DNA of DAS1529), molecularanalysis was carried out by Southern blot hybridization and PCR. For PCRvalidation, ORF6 (tccC-like) and ORF7 (Cry1529) specific primers(Example 8, Table 8) were used to amplify ORF6 and ORF7 from SB12 cosmidand DAS1529 total DNA. For ORF6, PCR amplifications were performed on aPE9600 thermal cycler (Perkin Elmer) with the following parameters:initial denaturation at 95° C. for 2 minutes; 30 cycles each withdenaturing at 95° C. for 30 seconds, annealing at 60° C. for 45 seconds,extension at 72° C. for 2 minutes, and a final extension for 10 minutesat 72° C. For ORF7, amplification parameters were the same as ORF6,except the annealing temperature was 55° C. for 30 seconds and extensionat 72° C. for 4 minutes. Specific PCR products with a single band ofexpected sizes were amplified for both ORF6 and ORF7.

[0261] Initial southern blot hybridization was based on partial SB12 DNAsequence and carried out according to standard protocol (Sambrock etal., 1990). DNA samples included total DNA of DAS1529 from twoindependent preparations, SB12 cosmid DNA, and one negative control DNAsample from NC1 (Photorhabdus). Both DAS1529 DNA samples were 16S rDNAsequence confirmed to be of Paenibacillus sp. origin, and one wasoriginally used for cosmid library construction; the other was a newpreparation. DNA samples were digested with EcoRI, blotted ontomembrane, and hybridized with Roche DIG System (Roche) labeled 180 bp ofPCR product amplified out of SB12. The PCR primers are 5′CCT CAC TAA AGGGAT CAC ACG G 3′ annealing partially to the vector and truncated ORF1(compared to full-length ORF3), and 5′ GGC TAA TTG ATG AAT CTC CTT CGC3′ annealing to the truncated ORF1 (tcaA-like) and full length ORF3(tcaA-like). A total of three DNA fragments (0.85, 2.7, and 8.0 kb)hybridizing to the PCR probe were detected, 0.85 and 8.0 in the SB12 and2.7 and 8.0 in DAS1529 DNAs. No signals were detected in the negativecontrol. The 0.85 kb (from first EcoRI ORF1 internal fragment to firstEcoRI site in the vector) and 8.0 kb (from first 5′ EcoRI site in ORF3to the third EcoRI site in ORF1) matched the calculated sizes of thetarget DNA fragments from SB12. Detection of the 2.7 kb fragmentsuggests the presence of an EcoR1 site 2.7 kb immediately upstream ofthe first EcoRI site within ORF1 in DAS1529 DNA. Those results show thatthe SB12 insert was from DAS1529 total DNA and, based on hybridizationand restriction analysis, all copies of the ORFs were accounted for.

EXAMPLE 8 Characterization of Insecticidal Activities of ProteinsEncoded by SB12 Cosmid ORFs

[0262] Random transposon insertional mutagenesis (to disrupt anindividual ORF or an entire operon) and heterologous expression(expressing individual ORFs or entire operons) were undertaken toisolate individual ORF(s) or operons conferring the insecticidalactivities in the SB12 cosmid.

[0263] A. Random Transposon Mutagenesis of SB12 Cosmid

[0264] A Tn mutagenesis library was generated from DAS1529 cosmid SB12using the GPS-1 Genome Priming System (New England BioLabs, Beverly,Mass.) following the kit instructions. Briefly, 2 μl 10× GPS buffer, 1μl pGPS2.1 Donor DNA (0.02 μg), 1 μl SB12 cosmid (0.1 μg) and 18 μlsterile H₂O were added to a 0.5 ml tube. One μl of TnsABC Transposasewas added; the mixture was vortexed and then spun briefly to collect thematerials at the bottom of the tube. This reaction mixture was incubatedfor 10 minutes at 37° C. One μl of Start Solution was added and mixed bypipetting up and down several times. The reaction was incubated at 37°C. for 1 hour and was then heat inactivated at 75° C. for 10 minutes.One μl of the reaction mixture was diluted 10-fold with sterile H₂O; 1μl of the diluted reaction was electroporated into 100 μl of Electro MAXDH5α-E E. coli (Gibco BRL, Rockville, Md.). After 1 hour of outgrowth inSOC medium at 37° C., 10 μl or 100 μl were plated on LB agar platescontaining 20 μg/ml Kanamycin and 15 μg/ml chloramphenicol, followed byincubation overnight at 37° C.

[0265] Individual colonies from the SB12 Tn mutagenesis were streakedonto fresh LB agar plates containing 20 μg/ml Kanamycin and 15 μg/mlchloramphenicol. From the streaks, 50 ml cultures of LB containing 20μg/ml Kanamycin and 15 μg/ml chloramphenicol were inoculated and grownat 28° C., 200 rpm for 48 hours. The cells were then collected bycentrifugation at 3500 rpm for 20 minutes. The supernatant was removedand the pellet resuspended in 2.5 ml of the culture supernatant for a20× concentration. The concentrated cell pellet was then assayed foractivity against corn earworm. Forty μl of the 20× concentrate wassurface applied to artificial diet using 8 wells per sample in 128 wellplates. Newly hatched corn earworm larvae were added and allowed to feedfor 5 days, at which time mortality and weights were recorded.

[0266] A total of 184 clones were tested for loss of activity againstcorn earworm. The results are summarized in Table 7. Bioassay of Tnclones revealed that a Tn insertion in the Cry1529 gene results incomplete loss of activity. Initial bioassay showed that the activitiesof clones which carried Tn insertions in the tc genes were variable.Further analysis of those clones in which cultures were all normalizedto the same cell density prior to bioassay showed no loss of activity ascompared to SB12. Results from Tn analysis suggest that ORF7(Cry1529) isthe key insecticidally active component of SB12 cosmid. TABLE 7 Bioassayof SB12, Cry1529 and tc tn insertion E. coli Clones Insects TBW CEW THWGrubs SCRW SB12 +++ ++ +++ − n.d Tn in Cry1529 − − − − − Tn in tcs +++++ +++ − −

[0267] B. Heterologous Expression SB12 ORFs/Operon.

[0268] Cry1529 (ORF7) and five tc ORFs (see Table 8 below) wereexpressed in pET101D® system. See FIG. 5. This expression vector has allthe attributes of the basic T7-regulated pET expression system(Dubendorff and Studier, 1991; Studier and Moffatt, 1986) and allowsdirectional cloning of a blunt-end PCR product into a vector forhigh-level, regulated expression, and simplified protein purification inE. coli. Optimal PCR amplification employed high-fidelity PfuTurbo™ DNApolymerase that is highly thermostable and possesses a 3′ to 5′exonuclease proofreading activity to correct nucleotide-misincorportaionerrors (Stratagene, La Jolla, Calif.). When ThermalAce™ polymerase(Invitrogen) is used, point mutations were introduced in the tc ORFs,which were corrected by the PfuTurbo™ based Quick-Change™ XLsite-directed mutagenesis kit (Stratagene). The E. coli strain BL21Star™ (DE3), was used as a host for expression since it contains therne131 mutation (Lopez et al., 1999) that generally enhances mRNAstability and the yield of the recombinant proteins.

[0269] Individual ORFs were PCR amplified out of the SB12 cosmid withORF specific primers (Table 8) under defined conditions. As adirectional cloning requirement, the forward PCR primers were designedto contain the sequence, CACC, at the 5′ end to ensure PCR product basepair with the overhang sequence, GTGG, in the pET101.D vector. Thereverse primers when paired with forward primers will amplify each ORF,respectively. PCR reactions were carried out in 50 μl reaction mixturecontaining of 50 ng of SB12 cosmid DNA, 1× Pfu reaction buffer(Stratagene), 0.2 mM each of dNPT, 0.25 mM of each primer, and 2 U ofPfuTurbo DNA polymerase (Stratagene). PCR amplifications were performedon a PE9600 thermal cycler (Perkin Elmer) with the following parameters:initial denaturation at 95° C. for 2 minutes, 35 cycles each withdenaturing at 95° C. for 30 seconds, annealing at 55° C. for 30 seconds,extension at 72° C. for 2 minutes per kb ORF, and a final extension for10 minutes at 72° C. TABLE 8 Summary of PCR Primers for Cloning ORF1-7ORFs Forward primers (5′ to 3′) Reverse primers (5′ to 3′) ORF1 (tcaA₁)CACCATGCTTTATAAGGCCTGGC TCAGGCCTGCACCGC ORF3 (tcaA₂)CACCATGGTGTCAACAACAGACAACAC TCAGGCTTTCGCTGCAGC ORF4 (tcaB₂)CACCATGACCAAGGAAGGTGATAAGC CTATTTCATAACATATCGAATTGG ORF5 (tcaC)CACCATGCCACAATCTAGCAATGC TCACCGCGCAGGCGGTGAAG ORF6 (tccC)CACCATGAAAATGATACCATGGACTCATC CTACTTTCTCTTCATTGAAAACCGGCGG ORF7CACCATGAACTCAAATGAACCAAATTTATC AACTGGAATTAACTTCGATTC (Cry1529)

[0270] PCR products for each ORF were cloned into pET101.D followinginstructions from the supplier (Invitrogen). The cloned ORF was purifiedas pET101.D plasmid DNA and sequenced verified. Since Tn analysisindicated ORF7 is the key component of SB12 for control of the testedpests, biochemical analysis and insect bioassay focused onheterologously expressed ORF7 proteins. For ORF7 expression clones, DNAsequence analysis showed 100% match with the original SB12 DNA sequence.Expression of ORF7 was induced by 0.5 mM IPTG for 4 hrs according to kitinstructions (Invitrogen).

[0271] C. Bioassay for Insecticidal Activities of ORF7 and tc Operon.

[0272] Bioassay samples were prepared as whole E. coli cells, celllysates, and purified toxins. The spectrum and specific activity of ORF7(Cry1529) is summarized in Table 10. Cry1529 is most active againsttobacco hornworm (Manduca sexta) and highly active (LC50 of 10 μg/mldiet) against tobacco budworm (Heliothis virescens); 100% mortality wasobserved for both insects. At higher doses, Cry1529 conferred somemortality (20 to 60%) and substantial growth inhibition on corn earworm(Heliothis zea), beet armyworm (Spodoptera exigua), and black cutworm(Agrotis ipsilon). For European comborer (Ostrinia nubilalis), Cry1529had some growth inhibition at higher doses. For some other insectspecies (fall armyworm, boll weevil, southern rootworm, mosquito), noactivity was detected. The Cry1529 LC50s for Cry1A (Cry1Ac) resistantdiamond back moth (DBMr) and sensitive diamond back moth (DBM) coloniesare >50 μg/ml and <1.0 μg/ml, respectively, suggesting a potential crossresistance. Cry1529 did not confer detectable activity on grass grubs, arelative of Japanese beetles.

[0273] To test the activity of other non-Cry1529 factors in DAS1529, oneCry1529 tn knockout SB12 cosmid clone (tn67) was assayed against TBW,CEW, SCRW, ECB, BW, BAW, THW, and grass grubs; no activity was foundagainst these pests. To address the issue of potential non- orlow-expression of tc ORFs in SB12 background, individually expressed tcORFs were tested independently and in combination with the other TCsfrom DAS 1529; no activity was found against TBW, CEW, and grass-grubs.Further, four ORFs were expressed as a single operon to very high levelsin E. coli cells. When tested in vitro, the whole cells contained nodetectable activity on TBW, CEW, and grass-grubs. While the lack of grubactivity is somewhat interesting, these results are not surprising inthat Paenibacillus typically infect a narrow range of grub hosts. Inlight of this, it could follow that the spectrum of activity of theinsecticidal toxins might also be relatively narrow. Thus, screens(using known methods) involving a broader range of pests, and additionaltime, would be required to identify susceptible pests. The resultspresented herein should not lead one away from recognizing that thesubject TC proteins have utility as do other TC proteins fromXenorhabdus, Photorhabdus, and the like.

[0274] Soluble proteins were extracted with 25 mM sodium phosphate pH8.0, 100 mM sodium chloride and analyzed on 4-12% NuPAGE gradient gelwith 1× MES buffer (Invitrogen). ORF7 protein was purified usingstandard procedures, and N-terminal sequencing revealed the expectedsequence: MNSNEPNLSDV. A bioassay was performed with whole E. colicells, with normalized cell density, expressing target proteins. SeeFIG. 6. Large scale purified ORF7 protein was used to obtain LC50s forORF7 by in vitro bioassay. Thermal stability analysis of the purifiedORF7 indicated that a 5 minute treatment at 75° C. is sufficient toabolish its activity against TBW. See Table 9. TABLE 9 Thermal Stabilityof Purified Cry1529 (ORF7) Samples Activity Cry1529, room temperature+++ Cry1529, 50° C. for 5 min. +++ Cry1529, 50° C. for 10 min. +++Cry1529, 75° C. for 5 min. − Cry1529, 75° C. for 10 min. − Cry1529, 100°C. for 5 min. −

[0275] For the tc genes, error-free clones of ORF3 and ORF6 were used asintermediate clones to generate a tc operon clone expressing ORF3(tcaA), ORF4 (tcaB), ORF5 (tcaC), and ORF6 (tccC). To construct the tcoperon in pET101.D, the NsiI/SacI fragment containing partial tcaA,entire tcaB and tcaC, and partial tccC was excised out of SB12 cosmid toreplace the NsiI/SacI insert in pET101.D-tcaA; this was followed by theinsertion of a 208 bp SacI fragment from pET101.D-tccC. See FIG. 5. Allfour ORFS were expressed to high levels by standard IPTG induction. Forthe ORF6 (tccC) expressed in the tc operon, the size of the expressedprotein was slightly smaller than the ORF6 predicted by Vector NTI fromthe 5′-most ATG (SEQ ID NO:18) and expressed independently. Hence, theannotated ORF6 (SEQ ID NO:13) based on the presence of a ribosomebinding site consensus is likely the native protein produced in SB12 andDAS1529.

[0276] D. Activity Spectrums of Toxins

[0277] The toxin activity spectrum of Cry1529 (ORF7) is summarized inTable 10. TABLE 10 Spectrum activity for E. coli and Pseudomonasexpressed Cry1529 Material Active Production Species (+++) Format &Method Method LC₅₀ H. virescens (TBW) +++  96 well top load and dietFCP, SE, 11 μg tox/ml diet with incorporation (scores, purified, IC E.coli cell preps mortality, inhibition) H. zea (CEW) +  96 well top loadand diet FCP, SE, >100 μg tox/g diet incorporation (scores, purifed, ICmortality, inhibition) S. exigua (BAW) +  96 well top (score) FCP,purifed >78 μg/cm² S. frugiperda (FAW) −  96 well top (score) FCP,purifed >>10 μg/cm² Plutella xylostella (DBM) +++  96 well top (score)FCP, purifed 0.02 μg tox/g diet Cry1 A resistant Plutella +  96 well top(score) FCP, purifed 59.7 μg tox/g diet xylostella (rDBM) A. ipsilon(BCW) +  96 well top (score) FCP, purifed >10 μg/cm² O. nubilalis(ECB) + 128 well top (weights) FCP, purifed >43 μg/cm² Culex sp.(Mosquito) − 1 oz cups (mortality) FCP, purifed >20 μg/ml H₂0 Diabroticaundecimpunctata −  96 well top (score) FCP, purifed >>100 μg tox/cm²howardi (SCRW) Anthonomous grandis − 128 well top (weights) FCP,purifed >>43 μg tox/cm² grandis (BW) M. sexta (THW) +++ (highly active)Continis mutabilis (Beetles); − >>100 μg tox/g soil surrogate for grassgrub

[0278] Only a limited range of pests was used in assays in an initialattempt to determine the activity spectrum of the subject TCs/tc ORFs.The following data, using the ORF3-OR6 operon, were obtained: TABLE 11Spectrum activity for Tc ORF's Format Material Active & High ProductionSpecies (+++) Method Dose Method Comments H. virescens − 96 well 10× FCPNo effect (TBW) top (score) H. zea − 96 well 10× FCP No effect (CEW) top(score) S. exigua − 96 well 10× FCP No effect (BAW) top (score)Spodoptera 96 well 10× FCP No effect frugiperda top (FAW) (score) A.ipsilon − 96 well 10× FCP No effect (BCW) top (score)

[0279] Again, while this initial round of screening did not revealactivity of these TCs against these pests, one skilled in the art wouldnot doubt that the subject proteins are useful, as are the correspondingPhotorhabdus/Xenorhabdus proteins. In addition, see Example 10, below.

EXAMPLE 9 Use of PCR Primers for Identifying Cry1529 Homologues fromOther Bacterial Genera, Species, and Strains

[0280] For screening additional ORF7 cry1529 homologs from other(Paenibacillus or other) strains, gene specific and degenerate PCRprimers were designed to amplify the target ORF7 DNA sequences of 1 kb.The PCR primers were deduced from two, well-conserved protein motifs(QAANLHL, domain I, block 1 core for forward primer; GPGFTGGD, domainIII, block 3 for reverse primer) highly conserved in Cry proteins. Thoseprimers are listed in Table 12 and were validated on DAS1529. PCRamplifications were performed on a PE9600 thermal cycler (Perkin Elmer)with the following parameters: initial denaturation at 95° C. for 2minutes; 35 cycles each with denaturing at 95° C. for 30 seconds,annealing at 47° C. for 45 seconds, extension at 72° C. for 2 minutes,and a final extension for 10 minutes at 72° C. Those primer pairs wereused to screen a bacterial (non-B. thuringiensis) culture collection byPCR. Five out of 192 strains (three Paenibacillus, one Bacillus, and oneunidentified) produced PCR products of expected sizes. These strainswere also found to have CEW activity according to primary bioassayscreening. However, sequence analysis of amplicons obtained from one ofthese strains using different primers showed that the amplicons were notderived from a cry gene.

[0281] Notwithstanding this, and as these screens were not exhaustive,the subject invention includes methods of screening Paenibacillus spp.,Bacillus spp. (including Bacillus thuringiensis and sphaericus), and thelike for Cry1529-like proteins and genes. Given the significant natureof the discovery of lepidopteran-toxic Cry proteins in Paenibacillus,the subject invention also includes methods of screening Paenibacillusspp., generally, for lepidopteran-toxic Cry proteins and genes. Variousscreening methods are well-known in the art, including PCR techniques(as exemplified above), probes, and feeding assays (where whole cellsare fed to target pests). As one skilled in the art would readilyrecognize, screening methods of the subject invention include thepreparation and use of clone libraries (such as cosmid libraries) inthese screens. TABLE 12 PCR Primers for Screening ORF7 HomologsGene-specific and degenerate DNA sequence (5′ to Primers 3′) Cry1529-FCAAGCAGCCAACCTCCACCTA Cry1529-R ATCCCCTCCTGTAAAGCCTGG CryPP-FCAAGCNGCNAATYTWCATYT CryPP-R TCNCCNCCNGTAAANCCWGG CryPT-FCARGCSGCSAAYYTBCAYYT CryPP-F2 CAAGCWGCWAATYTWCATYT CryPP-R2TCHCCWCCWGTAAAWCCWGG CryPT-F2 CAGGCSGCSAAYYTGCATYT

EXAMPLE 10 Complementation of Xenorhabdus XptA2 TC Protein Toxin withDAS1529 TC Proteins

[0282] This example provides experimental evidence of the ability ofDAS1529TC proteins, expressed here with a single operon (ORFs 3-6; tcaA,tcaB, TcaC and tccC; see section C of Example 8), to complement theXptA2 toxin from Xenorhabdus nematophilus Xwi (see SEQ ID NO:49). Twoindependent experiments were carried out to express the DAS1529 TCoperon and XptA2 independently, or to co-express the XptA2 gene and theTC operon in the same E. coli cells. Whole cells expressing differenttoxins/toxin combinations were tested for activity against thelepidopteran insects: corn earworm (Heliothis zea; CEW) and tobaccobudworm (Heliothis virescens; TBW). The data from both experimentsindicate that DAS1529 TC proteins are able to enhance Xenorhabdus TCprotein XptA2 activity on both insect species tested.

[0283] A. Co-expression of DAS1529 TCs and Xenorhabdus XptA2

[0284] Expression of the TC operon was regulated by the T7 promoter/lacoperator in the pET101.D expression vector that carries a ColE1replication origin and an ampicillin resistance selection marker(Invitrogen). Comprehensive description of cloning and expression of thetc operon can be found in section C of Example 8. The XptA2 gene wascloned in the pCot-3 expression vector, which carries a chloramphenicolresistance selection marker and a replication origin compatible with theColE1. The pCot-3 vector expression system is also regulated by the T7promoter/lac operator. Hence, compatible replication origins anddifferent selection markers form the basis for co-expression of the TCoperon and XptA2 in the same E. coli cells. Plasmid DNAs carrying the TCoperon and XptA2 were transformed into E. coli, BL21 Star™ (DE3) eitherindependently or in combination. Transformants were selected on LB agarplates containing 50 μg/ml carbenicillin for pET101.D-TC operon, 50μg/ml chloramphenicol for pCot-3-XptA2, and both antibiotics forpET101.D-TC operon/pCot-3-XptA2. To suppress basal toxin expression,glucose at a final concentration of 50 mM were included in both agar andliquid LB medium.

[0285] For toxin production, 5 ml and 50 ml of LB medium containingantibiotics and 50 mM glucose were inoculated with overnight culturesgrowing on the LB agar plates. Cultures were grown at 30° C. on a shakerat 300 rpm. Once the culture density has reached an O.D. of ˜0.4 at 600nm, IPTG at a final concentration of 75 μM was added to the culturemedium to induce gene expression. After 24 hours, E. coli cells wereharvested for protein gel analysis by the NuPAGE system (Invitrogen).Cell pellets from 0.5 ml 1× culture broth was resuspended in 100 μl of1× NuPAGE LDS sample buffer. Following brief sonication and boiling for5 min, 5 μl of the sample was loaded onto 4 to 12% NuPAGE bis-trisgradient gel for total protein profile analysis. XptA2 expressed todetectable levels when expressed independently or in the presence of theTC operon. Based on gel scan analysis by a Personal Densitometer SI(Molecular Dynamics), XptA2 expressed nearly 8× as high by itself aswhen co-expressed with the TC operon. For the 5 ml induction experiment,there is a nearly equal expression of XptA2.

[0286] B. Bioassay for Insecticidal Activity

[0287] As described in Example 8, DAS1529 tc ORFs when expressedindependently or as an operon, did not appear to be active against TBWand CEW. The following bioassay experiments focused on determiningwhether Paenibacillus (DAS1529) TC proteins (of ORFs 3-6; TcaA-, TcaB-,TcaC-, and TccC-like proteins) can complement Xenorhabdus TC proteintoxin activity (XptA2 is exemplified). Bioassay samples were prepared aswhole E. coli cells in 4× cell concentrate for the 5 ml inductionexperiment, both the XptA2 and XptA2/TC operon cells contained very lowbut nearly equal amount of the XptA2 toxin. Data in Table 13 showed thatat the 4× cell concentration tested, TC proteins+Xenorhabdus XptA2 wasactive against CEW. This provided the first evidence of acomplementation effect of Paenibacillus DAS1529 TC proteins onXenorhabdus XptA2. TABLE 13 Bioassay of DAS1529 TC complementation ofXeno. XptA2 on H. zea Insects: CEW Negative control − TCs (DAS1529) −Xeno. XptA2 − TC proteins + Xeno. XptA2 ++

[0288] For the second bioassay experiment, the amount of XptA2 proteinin the XptA2 cells and the XptA2+TC operon cells was normalized based ondensitometer gel scan analysis. As shown in Table 14, XptA2 per se hadmoderate activity at 40× on TBW (H. virescens), but the activity droppedto a level undetectable at and below 20×. However, when co-expressedwith TCs, high levels of activity were very apparent in the presence of10× and 5× XptA2, and low activity was still noticeable at 1.25× XptA2.These observations indicate there is a significant potentiation effectof 1529 TC proteins on Xenorhabdus XptA2 against H. virescens. At thehighest doses tested, neither the negative control nor the tc operon perse had any activity against this pest. TABLE 14 Bioassay of IDAS1529 TCcomplementation of XptA2 on H. virescens Normalized XptA2 40× 20× 10× 5×2.5× 1.25× XptA2 + − − − n.d. n.d. TCs + n.d. n.d. ++ ++ + − XptA2

EXAMPLE 11 Stabilization of Cry1529 Protein Against Trypsin Digestion

[0289] This example teaches modifications to the DNA sequence disclosedas SEQ ID NO:14, which encodes the Cry1529 protein (disclosed as SEQ IDNO:15) such that the new encoded proteins are more resistant toproteolytic digestion by trypsin than is the native protein. Digestionof proteins in the gut of insects limits the time of exposure of theinsect to a protein toxin. Therefore, methods that decrease thesusceptibility of a protein toxin to protease digestion can be used toincrease potency of the protein.

[0290] For these tests, trypsin enzyme (e.g. Sigma Chemical #T1426) andtrypsin inhibitors (e.g. Sigma Chemical #T9008) were prepared as stocksolutions of 4 mg/mL or 10 mg/mL in 50 mM Tris HCl buffer, pH8.0. Testincubations with various concentrations of trypsin and Cry1529 proteinwere performed at 37° C. for 1 hour, and were terminated by addition ofan equal volume of an equal concentration of trypsin inhibitors (e.g. adigestion that received 35 μL of 4 mg/mL trypsin solution was terminatedby addition of 35 μl of 4 mg/mL trypsin inhibitors). For a typicalexperiment, Cry1529 protein was produced by appropriately engineered E.coli cells and purified by steps described previously, which includedseparation from other proteins by passage through a size-exclusioncolumn. Following digestion, the protease products were analyzed bystandard acrylamide gel electrophoresis followed by immunoblot analysisusing antibody prepared against the Cry1529 protein. The results of suchan experiment are shown in FIG. 9.

[0291] Trypsin digestion produces two major protein products, thesmaller of which is approximately 50 kDa in molecular size. It is notedthat this digestion pattern is the same as that produced from trypsindigestion of a Cry1529-His₆ protein, which is identical to the nativeCry1529 protein amino acid sequence of SEQ ID NO:15 except for theaddition of amino acids KGELNSKLEGKPIPNPLLGLDSTRTG HHHHHH to thecarboxy-terminus. The coding region for Cry1529-His₆ was produced byligating the coding region for the native Cry1529 protein into thepET101/D-TOPO® vector (Invitrogen™, Carlsbad, Calif.). This recombinantclone was made to facilitate purification of the recombinant Cry1529protein by binding to a commercially available V5 antibody, whoseepitope is represented by the amino acid sequence GKPIPNPLLGLDSTRTG(underlined above), or by purification schemes that expoit the sixhistidine residues (double underlined above). Procedures for thesemanipulations were performed according to the recommendations providedwith the pET101/D-TOPO® vector.

[0292] Trypsin digestion of the Cry1529-His₆ protein was found toeliminate activity in insect bioassays against lepidopteran insects.MALDI-TOF analysis was used to determine the sequence of amino acidscomposing the N-terminus of the 50 kDa peptides, and two proteaseprocessing sites were determined, corresponding to amino acid residues122 (R, Arginine) and 126 (K, Lysine) of SEQ ID NO:15.

[0293] Modifications to remove the first trypsin cleavage site in theencoded protein were made in the native DNA sequence (SEQ ID NO:14),using the QuickChange® mutagenesis methodology (Stratagene, La Jolla,Calif.). Three different types of mutations were made at amino acids inthe region of 120 to 123 of SEQ ID NO:15: RARA to HANA, RARA to RARS,and RARA to QANA. The DNA oligonucleotide primers (listed in the 5′ to3′ direction for each strand) for these mutations are listed in Table 15below. The bases that differ from the native DNA sequence areunderlined. TABLE 15 Reverse (Complementary Mutation Forward (Codingstrand) Primer strand) Primer RARA to HANA AAAATGATTCTAATAATTTACACGCGAACGTCTTTCACTACAGCGTTCGCGTGTAAATTA (pMYC2865) GGTGTAGTGAAAGAC TTAGAATCATTTTRARA TO QANA AAAATGATTCTAATAATTTACAAGCGAACGTCTTTCACTACAGCGTTCGCTTGTAAATTA (pMYC2866) GCTGTAGTGAAAGAC TTAGAATCATTTTRARA TO RARS AAAATGATTCTAATAATTTAAGAGCGAGAGTCTTTCACTACAGATCTCGCTCTTAAATTA (pMYC2867) TCTGTAGTGAAAGAC TTAGAATCATTTT

[0294] Comparison of the wild type and mutated coding regions induced bythese primers are shown in this Table. The pertinent amino acid residuesare shown in bold type. TABLE 16 Wild-type: gAA AAT GAT TCT AAT AAT TTAAGA GCG AGA GCT GTA GTG AAA GAC Amino Acids: (E) N   d   s   n   n   l   R    A   R   A    V   V   K   D        115                 120     122 123     125 126 RARA to HANA:gAA AAT GAT TCT AAT AAT TTA CAC  GCG AAC  GCT GTA GTG AAA GAC AminoAcids: (E)  N   D   S   N   N   L   H    A   N    A   V   V   K   D RARAto QANA: gAA AAT GAT TCT AAT AAT TTA CAA  GCG AAC  GCT GTA GTG AAA GACAMINO ACIDS: (E)  N   D   S   N   N   L   Q    A   N   A   V   V   K   D RARA to RARS: gAA AAT GAT TCT AAT AAT TTA AGA GCGAGA TCT  GTA GTG AAA GAC Amino Acids:(E)  N   D   S   N   N   L   R   A   R   S    V   V   K   D

[0295] The separate, mutated coding regions were each cloned into thepET101/D-TOPO® vector, which allows inducible production of the Cry1529variant proteins. E. coli cells containing the constructs were grown,and expression of the Cry1529 variant genes was induced by methodsrecommended by the supplier. Harvested whole cells were then tested intrypsin digestion assays, and analyzed as above. Typical results areshown in FIG. 10. For these experiments, 10 mg of whole cell pellet wassuspended in 50 mM Tris HCl, pH8.0, and digested for 3 hours at 37° in afinal volume of 1 mL, with 100 μL of 10 mg/mL trypsin. The reactionswere mixed occasionally during incubation. Digestion was terminated byaddition of 100 μL of 10 mg/mL trypsin inhibitors and the tubes werestored on ice.

[0296] These results demonstrate that both the native Cry1529 (RARA) andthe Cry1529-His₆ (RARA) proteins are digested by trypsin to produce amajor product of about 50 kDa. When the RARA sequence corresponding tothe trypsin cleavage site was mutated to HANA or QANA, substantialresistance to trypsin digestion was obtained. No 50 kDa peptides wereproduced, and easily detectable amounts of the apparently full-lengthCry1529-His₆ proteins were present. Mutation of the RARA site to RARSdid not eliminate production of the 50 kDa peptides, but substantiallyreduced the rate of protease cleavage. Thus, it is apparent thatmutation of protease processing sites in the Cry1529 proteinsubstantially decreases its susceptibility to protease digestion. Thisallows the proteins to reside for longer periods of time in the insectgut following ingestion, resulting in increased potency to killsusceptible insects.

EXAMPLE 12 Design of PCR Primers for Detection of Homologues of IDAS1529 tcORFs in other Paenibacillus Strains

[0297] As shown above, Paenibacillus strain IDAS 1529 produces anextracellular protein that is toxic to various Lepidopteran insects.Molecular phylogeny of the 16S ribosomal gene of this strain indicatesthat it is most closely related to members of the P. thiaminolyticus-P.lentimorbus-P. popilliae cluster. It has also been shown thatPaenibacillus strain IDAS 1529 contains both toxin complex genes(hereafter designated as tc genes) and a novel insecticidal crystallineinclusion protein gene designated cry1529. In an attempt to determine iftc homologues are present in other members of the genus Paenibacillus, acollection of Paenibacillus strains was screened by polymerase chainreaction (PCR) and hybridization analyses. For the PCR analyses, totalDNA isolated from Paenibacillus strains was used as template andscreened using oligonucleotide primers specific to tc genes found inPaenibacillus strain IDAS 1529, Photorhabdus species, and Xenorhabdusspecies. Amplified products obtained with the tc primer sets were clonedand their nucleotide sequence was determined and compared to tcsequences obtained from Paenibacillus strain IDAS 1529. The followingExamples illustrate how one can design tc-specific oligonucleotideprimers and use PCR to search the total DNA of Paenibacillus isolatesfor DNA sequences that are homologous to tc genes identified inPaenibacillus strain IDAS 1529, Photorhabdus species, and Xenorhabdusspecies. By using PCR analysis (as described herein), it was (and is)possible to identify tc homologues in a species of Paenibacillusdistinct from Paenibacillus strain IDAS 1529 and the P.thiaminolyticus-P. lentimorbus-P. popilliae cluster.

[0298] 12.A.—Extraction of Total DNA from Paenibacillus Strains

[0299] Paenibacillus strains were grown on nutrient agar plates (8 g/lnutrient broth, 15 g/l Bacto agar; Difco Laboratories, Detroit, Mich.)for 3-5 days at 30° C. A single colony was picked and inoculated into a500 ml tribaffled flask containing 100 ml of sterile nutrient broth (8g/l nutrient broth; Difco Laboratories, Detroit, Mich.). Following 24-72hrs of incubation at 30° C. on a rotary shaker at 150 rpm, the cultureswere dispensed into sterile 500 ml polyethylene bottles and centrifugedat 6, 500×g for 1 hr at 4° C. After centrifugation, the supernatantfluid was decanted and the bacterial cell pellet was retained. Total DNAwas extracted from the cell pellet using the QIAGEN Genomic-tip System100/G and associated Genomic DNA Bufffer Set (QIAGEN Inc., Valencia,Calif., USA) by following The Sample Preparation and Lysis Protocol forBacteria exactly as described by the manufacturer. The extracted totalDNA was solubilized in 0.5 ml TE buffer (10 mM Tris-HCl, pH 8.0; 1 mMEDTA, pH 8.0).

[0300] 12.B.—Selection of tc Specific Oligonucleotide Primers for PCR

[0301] To select oligonucleotide primers specific to the tc genespreviously identified from Paenibacillus strain IDAS 1529, the tcaA,tcaB, tcdB and tccC nucleotide sequences obtained from Paenibacillusstrain IDAS 1529, Photorhabdus strain W 14, and Xenorhabdus strain Xwiwere aligned using the Align program in the Vector NTI software package(Informax, Inc., Frederick, Md.). Nucleotide sequences used for thisanalysis are listed in Table 17. TABLE 17 Nucleotide sequences used fortc specific primer selection Source of nucleotide Gene Gene Sourceorganism sequence Designation tcaA1 Paenibacillus strain SEQ ID NO: 2tcaA1-1529 IDAS 1529 tcaA2 Paenibacillus strain SEQ ID NO: 6 tcaA2-1529IDAS 1529 tcaA Photorhabdus strain W14 GenBank: tcaA-W14 Accession No.AF346497 tcaB1 Paenibacillus strain SEQ ID NO: 4 tcaB1-1529 IDAS 1529tcaB2 Paenibacillus strain ISEQ ID NO: 8 tcaB2-1529 IDAS 1529 tcaBPhotorhabdus strain W14 GenBank: tcaB-W14 Accession No. AF346497 tcdB1Photorhabdus strain W14 SEQ ID NO: 42 tcdB1-W14 tcdB2 Photorhabdusstrain W14 SEQ ID NO: 43 tcdB2-W14 xptC1 Xenorhabdus strain Xwi SEQ IDNO: 20 xptC1-Xwi tcaC Paenibacillus strain SEQ ID NO: 10 tcaC-1529 IDAS1529 tccC1 Photorhabdus strain W14 SEQ ID NO: 44 tccC1-W14 tccC2Photorhabdus strain W14 SEQ ID NO: 45 tccC2-W14 tccC3 Photorhabdusstrain W14 SEQ ID NO: 46 tccC3-W14 tccC4 Photorhabdus strain W14 SEQ IDNO: 47 tccC4-W14 tccC5 Photorhabdus strain W14 SEQ ID NO: 48 tccC5-W14xptB1 Xenorhabdus strain Xwi SEQ ID NO: 21 xptB1-Xwi tccC Paenibacillusstrain SEQ ID NO: 19 tccC-1529 IDAS 1529

[0302] 12.B.i.—tcaA Specific Primer Selection

[0303] Nucleotide sequence alignment of tcaA1-1529, tcaA2-1529, andtcaA- W14 identified two regions of nucleotide sequence identity ofsufficient length for the selection of PCR primers with minimaldegeneracy (shown as boxed regions in FIG. 10.). These two regions wereselected for the synthesis of tcaA specific primers, which weredesignated SB105 and SB106 (Tables 18 and 19).

[0304] 12.B.ii.—tcaB Specific Primer Selection

[0305] Nucleotide sequence alignment of tcaB1-1529, tcaB2-1529, andtcaB-W14 identified four regions of nucleotide sequence identity ofsufficient length for the selection of PCR primers with minimaldegeneracy (FIG. 11.). These four regions were selected for thesynthesis of tcaB specific primers, which were designated as SB101,SB102, SB103, and SB104 (Tables 18 and 19).

[0306] 12.B.iii.—tcaC Specific Primer Selection

[0307] Nucleotide sequence alignment of tcdB1-W14, tcdB2-W14, xptC1-Xwiand tcaC-1529 identified two regions of nucleotide sequence identity ofsufficient length for the selection of PCR primers with minimaldegeneracy (FIG. 12.). These two regions were selected for the synthesisof tcaC specific primers, which were designated as SB215 and SB217(Tables 18 and 19).

[0308] 12.B.iv.—tccC Specific Primer Selection

[0309] Nucleotide sequence alignment of tccC1-W14, tccC2-W14, tccC3-W14,tccC4-W14, tccC5-W14, xptB1-Xwi and tccC-1529 identified two regions ofnucleotide sequence identity of sufficient length for the selection ofPCR primers with minimal degeneracy (FIG. 13.). These two regions wereselected for the synthesis of tccC specific primers, which weredesignated as SB212 and SB213 (Tables 18 and 19). TABLE 18 tc specificprimers Primer Primer designation length Sequence of primer (5′ to 3′)SEQ ID NO. SB101 32 GCKATGGCSGACCCGATGCAWTACAAGCTGGC* 22 SB102 32AGCGGYTGACCRTCCAGRCTCARATTGTGGCG 23 SB103 28TGTATAACTGGATGGCYGGWCGTCTSTC 24 SB104 26 TCRAAAGGCAGRAAMCGGCTGTCGTT 25SB105 28 CTTCYCTKGATATCYTKYTGGATGTGCT 26 SB106 30ACGRCTGGYATTGGYAATCAGCCARTCCAA 27 SB212 27 CGYTATIAATATGAYCCKGTVGGYAAT28 SB213 25 CATCBCGYTCTTTRCCIGARTARCG 29 SB215 33CGHAGCTCYICCCAGTWYTGGCTGGATGARAAA 30 SB217 32GTRTCATTTTCATCTTCRTTBACIRYAAACCA 31

[0310] TABLE 19 tc primer combinations Target Forward ReverseApproximate size of expected amplified gene primer primer product tcaASB105 SB106  1.4 kb tcaB SB101 SB102  0.4 kb tcaB SB103 SB104 0.65 kbtcdB SB215 SB217  2.2 kb tccC SB212 SB213  0.9 kb

Example 13 PCR Amplification of Paenibacillus DNA

[0311] For PCR amplification using tcaA- and tcaB-specific primer sets,3-5 ul of total DNA obtained from each of the Paenibacillus strains wasmixed with 50 pmoles of each primer and 1× Eppendorf MasterMix(Eppendorf AG; Hamburg, Germany) in a 20 ul reaction volume.Amplification conditions were denaturation at 94° C. for 3 minutesfollowed by 30 cycles of denaturation at 94° C. for 1 minute, annealingat 52° C. for 1.5 minutes, and extension at 72° C. for 1.5 minutes,followed by a final extension at 72° C. for 5 minutes.

[0312] For PCR amplification using tcaC- and tccC-specific primer sets,approximately 375 ng of total DNA obtained from each of thePaenibacillus strains was mixed with 50 pmoles of each primer and 12.5ul of Epicentre® FailSafe™ Buffer D and 2.5 U of Epicentre® FailSafe™Polymerase (Epicentre; Madison, Wis.) in a 25 ul reaction volume.Amplification conditions were denaturation at 96° C. for 4 minutesfollowed by 40 cycles of denaturation at 94° C. for 30 seconds,annealing at 64° C. for 30 seconds, and extension at 70° C. for 30seconds. Each cycle, the annealing temperature was lowered by 0.5° C.and the extension time was increased by 5 seconds.

[0313] 13.A.—Gel Electrophoresis, Cloning, and Nucleotide SequenceDetermination of PCR Amplified Products

[0314] PCR amplification reactions were examined by gel electrophoresisusing 0.8 to 1% Seakem LE agarose (BioWhittaker Molecular Applications,Rockland, Me.) in 1× TAE buffer. Amplified products were cloned in thevector pCR 2.1 -TOPO® using the TOPO TA® Cloning Kit (Invitrogen™ LifeTechnologies, Carlsbad, Calif.) exactly as described by themanufacturer. The nucleotide sequences of the cloned amplified productswere determined using M13 Forward, M13 Reverse, and tc sequence-specificsequencing primers as needed to obtain double stranded sequence of eachcloned amplified product. Nucleotide sequencing was performed using theCEQ Dye Terminator Cycle Sequencing Quick Start Kit (Beckman Coulter,Fullerton, Calif., USA) and the CEQ 2000 XL DNA Analysis System (BeckmanCoulter) exactly as directed by the manufacturer. The Sequencher (v.4.1.4) software package (Gene Codes, Ann Arbor, Mich.) was used toconstruct contigs from the sequencing data and determine a consensussequence for each amplified product.

[0315] 13.B.—Nucleotide Sequence Analysis of PCR Amplified Products

[0316] 13.B.i.—tcaA

[0317] When PCR using the tcaA-(primer combination SB105 and SB106) wasperformed using total DNA obtained from the collection of Paenibacillusstrains, it was observed that total DNA from a Paenibacillus apiariusstrain (NRRL NRS 1438; hereafter designated as DB482) produced anamplified product of the expected sizes. The amplified product wascloned and sequenced.

[0318] The amplified product obtained using the SB105 and SB106 primercombination was designated as tcaA2-DB482. When the sequence oftcaA2-DB482 (SEQ ID NO:32) as compared to the tcaA sequences obtainedfrom Paenibacillus strain IDAS 1529 and Photorhabdus strain W14, it wasobserved that tcaA2-DB482 have the greatest nucleotide sequence identity(90.5% over 1,239 nucleotides) to tcaA2-1529 (Table 20). The deducedamino acid sequence encoded by tcaA2-DB482 (designated as TcaA2-DB482;SEQ ID NO:33) was 89.1% identical to the corresponding deduced aminoacid sequence of tcaA2-1529 (designated as TcaA2-1529; SEQ ID NO:7).TABLE 20 Nucleotide and deduced amino acid sequence identity oftcaA2-DB482 withcorresponding regions of tcaA1-1529, tcaA2-1529, andtcaA-W14 % deduced amino acid % Nucleotide identity with sequenceidentity with Gene tcaA2-DB482 tcaA2-DB482 tcaA1-1529 57 33 tcaA2-152990 89 tcaA-W14 50 32

[0319] 13.B.ii.—tcaB

[0320] The amplified products obtained using the SB101 and SB102 primercombination and the SB103 and SB104 primer combination were designatedas tcaB2a-DB482 and tcaB2b-DB482, respectively. When the sequences oftcaB2a-DB482 (SEQ ID NO:34) and tcaB2b-DB482 (SEQ ID NO:35) werecompared to the tcaB sequences obtained from Paenibacillus strain IDAS1529 and Photorhabdus strain W14, it was observed that both of thesesequences have the greatest nucleotide sequence identity to tcaB1-1529and tcaB2-1529 (Table 21). The nucleotide sequence identity oftcaB2a-DB482 and tcaB2b-DB482 to tcaB2-1529 was 92.6% and 89.8%,respectively. The deduced amino acid sequences encoded by tcaB2a-DB482(designated as TcaB2a-DB482; SEQ ID NO:36) tcaB2b-DB482 (designated asTcaB2b-DB482; SEQ ID NO:37) were 91.2% and 91.1% identical,respectively, to the corresponding deduced amino acid sequence oftcaB2-1529 (designated as TcaB2-1529; SEQ ID NO:9). TABLE 21 Nucleotideand deduced amino acid sequence identity of tcaB2a-DB482 andtcaB2b-DB482 with corresponding regions of tcaB1-1529, tcaB2-1529, andtcaB-W14 % Nucleotide % Nucleotide % deduced amino % deduced aminoidentity with identity with acid sequence with acid sequence with GenetcaB2a-DB482 tcaB2b-DB482 TcaB2a-DB482 TcaB2b-DB482 tcaB1-1529 93 93 9492 tcaB2-1529 93 90 91 92 tcaB-W14 63 57 59 57

[0321] 13.B.iii.—tcdB

[0322] When PCR using the tcaC-specific primer combination (SB215 andSB217) was performed using total DNA obtained from DB482 produced anamplified product of the expected size. The amplified product was clonedand sequenced.

[0323] The amplified product obtained using the SB215 and SB217 primercombination was designated as tcaC-DB482. When the sequence oftcaC-DB482 (SEQ ID NO:38) was compared to the tcaC sequences obtainedfrom Paenibacillus strain IDAS 1529, Xenorhabdus strain Xwi andPhotorhabdus strain W14, it was observed that tcaC-DB482 has thegreatest nucleotide sequence identity (93.5% over 2,091 nucleotides) totcaC-1529 (Table 22). The deduced amino acid sequence encoded bytcaC-DB482 (designated as TcaC-DB482; SEQ ID NO:39) was 91.1% identicalto the corresponding deduced amino acid sequence of tcaC-1529(designated as TcaC-1529; SEQ ID NO:11). TABLE 22 Nucleotide and deducedamino acid sequence identity of tcaC-DB482 corresponding regions ofxptC1-Xwi, tcdB1-W14, and tcdB2-W14, and tcaC-1529 deduced amino %Nucleotide sequence % acid sequence Gene identity with tcaC-DB482identity with TcaC-DB482 tcaC-1529 93 91 xptC1-Xwi 50 35 tcdB1-W14 50 36tcdB2-W14 50 36

[0324] 13.B.iv.—tccC

[0325] When PCR using the tccC-specific primer combination (SB212 andSB212) was performed using total DNA obtained from the collection ofPaenibacillus strains, it was observed that total DNA from DB482produced an amplified product of the expected size. The amplifiedproduct was cloned and sequenced.

[0326] The amplified product obtained using the SB212 and SB213 primercombination was designated as tccC-DB482. When the sequence oftccC-DB482 (SEQ ID NO:40) was compared to the tccC sequences obtainedfrom Paenibacillus strain IDAS 1529, Xenorhabdus strain Xwi andPhotorhabdus strain W14, it was observed that tccC-DB482 has thegreatest nucleotide sequence identity (93.7% over 858 nucleotides) totccC-1529 (Table 23). The deduced amino acid sequence encoded bytccC-DB482 (designated as TccC-DB482; SEQ ID NO:41) was 95.5% identicalto the corresponding deduced amino acid sequence of tccC-1529(designated as TccC-1529; SEQ ID NO:13). TABLE 23 Nucleotide and deducedamino acid sequence identity of tccC-DB482 corresponding regions ofxptB1-Xwi, tc-W14, tccC-1529, and tcc genes from Photorhabdus strain W14% deduced amino % Nucleotide sequence acid sequence Gene identity withtccC-DB482 identity with TccC-DB482 tccC-1529 94 96 xptB1-Xwi 54 45tccC1-W14 54 48 tccC2-W14 56 45 tccC3-W14 56 46 tccC4-W14 56 46tccC5-W14 54 44

[0327] 13.C.—Summary of PCR Analyses

[0328] This example (and other examples herein) illustrate methods fordesigning oligonucleotide primers based on tc genes from three genera ofbacteria, and that the use of these primers for PCR screening ofPaenibacillus strains can identify tc homologues present in thosestrains. DB482, which is an isolate of Paenibacillus apiarius (depositedas NRRL B-30670) that was isolated from honey bee larva, was shown tocontain homologues of tcaA, tcaB, tcaC, and tccC. The finding of thesetc homologues confirms that Paenibacillus strain IDAS 1529 is not uniquewithin the genus Paenibacillus with regard to possessing tc genes.Therefore, one skilled in the art can now use these and other methods toidentify other tc homologues in other species of Paenibacillus such asP. chondroitinus, P. alginolyticus, P. larvae, P. validus, P. gordonae,P. alvei, P. lentimorbus, P. popilliae, P. thiaminolyticus, P.curdlanolyticus, P. kobensis, P. glucanolyticus, P. lautus, P.chibensis, P. macquariensis, P. azotofixans, P. peoriae, P. polymyxa, P.illinoisensis, P. amylolyticus, P. pabuli, and P. macerans.

EXAMPLE 14 Detection of Homologues of IDAS 1529 tcORFS in otherPaenibacillus Strains by Southern Hybridization

[0329] This example illustrates how one can use radioactively labeledDNA fragments as probes to search the genomic DNA of Paenibacillusisolates for DNA sequences (preferably having some homology to the knowntcORFs first detected in IDAS 1529). The results demonstrate thatsequences homologous to two of the tcORFs are detected in aPaenibacillus apairius isolate, DB482.

[0330] Genomic DNA from various Paenibacillus strains (or from E. colito serve as a negative control) was prepared as described above inExample 12, and was digested with restriction enzyme to produce multiplefragments. A typical digestion contained 8 μg of DNA in a total volumeof 400 μL of reaction buffer as supplied by the manufacturer of the EcoRI enzyme (New England Biolabs, Beverly, Mass.). The reaction, containing200 units of enzyme, was incubated overnight at 37° C., then placed onice. Digested DNA was further purified and concentrated by addition of30 μL of 3M sodium acetate (pH5.2) and 750 μL of ice cold 100% ethanol,followed by centrifugation. The DNA pellet was washed twice with 70%ethanol, dried under vacuum, and resuspended in 50 μl of TE buffer [10mM Tris HCl, pH8.0; 1 mM ethylenediaminetetraacetic acid (EDTA)]. Analiquot was then analyzed by agarose gel electrophoresis for visualassurance of limit digestion. In a similar manner, DNA of IDAS 1529cosmid SB12 was digested with EcoR I, and was used as a positive controlfor the hybridization experiments.

[0331] EcoR I digested genomic DNA fragments to be blotted for Southernanalysis were separated by electrophoresis through 0.7% or 1.2% agarosegels in TEA buffer (40 mM Tris-acetate, 2 mM EDTA, pH8.0) (1 μgDNA/well). On each gel, lanes containing a 1 kb DNA Molecular WeightLadder (Invitrogen™, Carlsbad, Calif.) were used to provide molecularweight size standards. The 15 fragment sizes larger than 500 bp in thisladder (in kilobases) are: 12.2, 11.2, 10.1, 9.2, 8.1, 7.1, 6.1, 5.1,4.1, 3.1, 2.0, 1.6, 1.0, 0.52, and 0.50. The DNA in the gel was stainedwith 50 μg/mL ethidium bromide, the gel was photographed, and then theDNA in the gel was depurinated (5 min in 0.2M HCl), denatured (15 min in0.5M NaOH, 1.5M NaCl), neutralized (5 min in 0.2M HCl) and transferredto MAGNA 0.45 micron nylon transfer membrane (Osmonics, Westborough,Mass.) in 2× SSC (20× SSC contains 3M NaCl, 0.3M sodium citrate, pH7.0). The DNA was crosslinked to the membrane by ultraviolet light(Stratalinker®; Stratagene, La Jolla, Calif.) and prepared forhybridization by incubating at 60° C. or 65° C. for 1 to 3 hours in“Minimal Hybridization” solution [contains 10% w/v polyethylene glycol(M.W. approx. 8000), 7% w/v sodium dodecylsulfate; 0.6× SSC, 5mM EDTA,100 μg/ml denatured salmon sperm DNA, and 10 mM sodium phosphate buffer(from a 1M stock containing 95 g/L NaH₂PO₄.1H₂O and 84.5 g/L Na₂HPO₄.7H₂O)].

[0332] DNA fragments of the tcORFs for use as hybridization probes werefirst prepared by Polymerase Chain Reaction (PCR) using SB12 cosmid DNAas template (see previous examples). The forward and reverse primers forthese amplifications are listed (5′ to 3′ directions of the respectiveDNA strands) in Table 24, below (bases in capital letters correspond toprotein coding regions). Primer Set One is designed to amplify, fromSB12 cosmid DNA, a DNA fragment that includes all of tcORF5, which isdisclosed as SEQ ID NO:10, and which has some similarity to thePhotorhabdus tcaC gene (Table 6). Primer Set Two is designed to amplify,from cosmid SB12, a DNA fragment that encodes the protein disclosed asSEQ ID NO:19. This DNA fragment and the encoded protein are somewhatlonger than the DNA sequence of tcORF6 disclosed as SEQ ID NO:12, andthe encoded protein disclosed as SEQ ID NO:13. The proteins disclosed asSEQ ID NO:13 and SEQ ID NO:19 both have some similarity to the proteinencoded by the Photorhabdus tccC gene (Table 6). The amplified PCRproducts were cloned into the pCR®2.1-TOPO® cloning vector (Invitrogen™,Carlsbad, Calif.), and fragments containing the tcORFs were releasedfrom the resulting clones by restriction enzyme digestion (listed in theTable below), followed by purification from agarose gels using theGenElute™ Agarose Spin columns (Sigma Chemical Co, St Louis, Mo.).Recovered fragments were concentrated by precipitation using theQuick-Precip™ Plus Solution according to the supplier's instructions(Edge BioSystems, Gaithersburg, Md.). TABLE 2 PCR Primer Set One SB12tcORF5 (SEQ ID No.10) Forward Primer SB126*gtacgtcatctagaaaggagatataccATGCCACAATCTAGCAATGCCGATATCAAGCTATTGTCReverse Primer SB127*tgacatcggtcgacattattaCCGCGCAGGCGGTGAAGCAAATAATGATGAGTCCATGGTA Cut frompCR®2.1-TOPO® clone with SalI + XbaI + PvuI and purify 4,368 bp fragmentPCR Primer Set Two SB12 tcORF that encodes SEQ ID No. 19; encompassingtcORF6 (SEQ ID No. 12) Forward Primer SB128*, **gtacgtcaactagtaaggagatataccATGAAAATGATACCgTGGACTCAcCATTATTTGCTTCACCReverse Primer SB129*tgacatcgctcgagattattaCTTTCTCTTCATTGAAAACCGGCGGAAAAAGTTCCCA Cut frompCR®2.1-TOPO® clone with EcoRI + SphI/ + PvuI and purify 2,925 bpfragment

[0333] Radioactively labeled DNA fragments were prepared using the HighPrime Radioactive Labeling Kit (Roche Diagnostics, Mannheim, Germany)according to the supplier's instructions. Nonincorporated nucleotideswere removed by passage through a QIAquick® PCR Purification column(Qiagen, Inc. Valencia, Calif.) according to the manufacturer'sinstructions. Labeling of approximately 100 ng of DNA fragments by thesemethods resulted in specific activities of approximately 0.1 μCi/ng. Thelabeled DNA fragments were denatured by boiling for 5 minutes, thenadded to the hybridization blot in Minimal Hybridization solution andincubated overnight at 60° C. or 65° C. Loose radioactivity was removedfrom the blot by rinsing at room temperature in 2× SSC, then moretightly bound radioactivity was removed by washing the blot for at leastone hour at 60° C. or 65° C. in 0.3× SSC +0.1% sodium dodecylsulfate. Atleast two such washes were performed. The blot was placed on X-ray filmat −80° C. with two intensifying screens, and the exposed film wasdeveloped after 1 to 3 days exposure. Blots were stripped of hybridizedDNA fragments by boiling for 10 minutes in 0.3× SSC +0.1% SDS, andreused once or twice for subsequent hybridizations.

[0334] Distinct fragments that hybridized to probes derived from PrimerSets One and Two were observed in genomic DNA obtained fromPaenibacillus apairius strain DB482. The probe derived from Primer SetOne (primers SB126 and SB127), which detects sequences homologous to theIDAS 1529 tcORF5, hybridized to fragments of estimated sizes (inkilobases) of 20, 10.2, and 8.4. Within this range of molecule sizes,mobilities of DNA fragments can provide only estimations of truemolecular sizes. Signal intensity for the fragments estimated to be 20kb and 8.4 kb were much more intense than the signal intensity for thefragment estimated to be 10.2 kb. Since each of these fragments is atleast twice the size of the probe fragment (about 4.4 kb), oneexplanation for these results is that multiple copies of genes that aresimilar to the probe derived from IDAS1529 tc ORF5, and thus are similarto the Photorhabdus tcaC gene, are present in the genome ofPaenibacillus apairius strain DB482. However, other explanations forthis outcome are possible.

[0335] The probe derived from Primer Set Two (primers SB128 and SB129),which detects sequences homologous to the IDAS 1529 tcORF6 and itsflanking 5′ end sequences, hybridized to fragments of estimated sizes(in kilobases) of 7.8 and 4.5. Signal intensity for the fragmentestimated to be 7.8 kb was very much more intense than the signalintensity seen for the fragment estimated to be 4.5 kb. One explanationfor this result is that Paenibacillus apairius strain DB482 has a singlegene similar to the IDAS 1529 tcORF6 and its 5′ flanking sequences, andthus is similar to the Photorhabdus tccC gene, and that EcoR I cleavesthe gene into two fragments that have unequal portions of the DNAsequences comprising the gene. However, other explanations for thisoutcome are possible, including the presence of multiple genes withdifferent amounts of absolute homology to the probe.

[0336] These results (detection by PCR amplification followed by DNAsequence analyses) confirm the presence of relatives of the PhotorhabdustcaC and tccC genes in Paenibacillus apairius strain DB482.

EXAMPLE 15 Insecticidal Activity of DB482

[0337] Paenibacillus strain DAS1529 has been shown to produce anextracellular protein that is toxic to Lepidopteran insects and has alsobeen shown to contain a cry gene, designated as cry1529. As this strainproduces an extracellular insecticidally active protein andintracellular insecticidally active proteins, the subject inventionincludes screening other strains of Paenibacillus for extracellular(released into culture supernatant fluid) and/or intacellular(cell-associated) insecticidally active agents. This example illustrateshow one can produce fermentation broths of Paenibacillus strains, how toprocess these broths, and how to test samples derived from these brothsfor insecticidal activity.

[0338] 15.A. Production and Processing of Paenibacillus FermentationBroths

[0339] Paenibacillus strains were grown on nutrient agar plates (8 g/lnutrient broth, 15 g/l Bacto agar; Difco Laboratories, Detroit, Mich.)for 3-5 days at 30° C. A single colony was picked and inoculated into a500 ml tribaffled flask containing 100 ml of sterile modified trypticsoy broth (tryptone 10-g/l, peptone 7 g/l, soytone 3 g/l, KCl 5 g/l,K₂PO₄ 2.5 g/l; Difco Laboratories, Detroit, Mich.). Following 72 hoursof incubation at 28° C. on a rotary shaker at 150 rpm, the cultures weredispensed into sterile 500 ml polyethylene bottles and centrifuged at4,000×g for 45 minutes at 4° C. After centrifugation, the supernatantfluid was decanted and filtered through a 0.22 um membrane filter(Millipore Corporation, Bedford, Mass.). The culture filtrate was thenconcentrated 20× using a Centricon Plus-20 centrifugal filter devicewith a 5,000 molecular weight cutoff membrane by centrifuging at4,000×g. The bacterial cell pellet was resuspended in 10 mM potassiumphosphate buffer (pH=8). These samples were then tested in insectbioassay for insecticidal activities contained in the processedsupernatant and cell pellet samples.

[0340] 15.B. Insect Bioassay of Processed Supernatant and Cell Pellets

[0341] The insect species included in these assays were Diabroticaundecimpunctata howardi (Southern corn rootworm, SCR), Helicoverpa zea(corn earworm, CEW), and Heliothis virescens (tobacco budworn, TBW) Theartificial diet used to rear and bioassay SCR was described previously(Rose, R. L. and McCabe, J. M. 1973. J. Econ. Entomol. 66, 398-400).Standard artificial lepidopteran diet (Stoneville Yellow diet) was usedto rear and bioassay ECB, CEW, and TBW. Forty ul aliquots of theconcentrated supernatant or cell pellet samples were applied directly tothe surface of wells (˜1.5 cm²) containing the artificial diet. Treateddiet wells were allowed to air-dry in a sterile flow-hood, and each wellwas infested with a single, neonate insect hatched fromsurface-sterilized eggs. Assay trays were then sealed, placed in ahumidified growth chamber, and maintained at 28° C. for 3-5 days.Mortality and larval weight determinations were then scored. Eightinsects were used per treatment.

[0342] 15.C. Insecticidal Activity of DB482

[0343] Concentrated supernatant and cell pellets from strain DB482 hadinsecticidal activity against SCR, TBW, and CEW relative to controltreatments (Table 25.) It is possible that the insecticidal activityassociated with concentrated supernatants and cell pellets from DB482are the result of two different insecticidal factors, one that iscell-associated (i.e. Cry-like) and another that is released from thecells (i.e. TC-like). However, it is also possible that the insecticidalactivities from both the concentrated supernatant and cell pellets fromDB482 are the result of the same insecticidal factors being present inboth cellular fractions. TABLE 25 Insecticidal activity of DB482Concentrated Insects Tested Supernatant activity Cell pellet activitySCR +++* +++ TBW ++ ++ CEW +++ ++ Medium controls − −

[0344] 15.D. Summary of Insecticidal Activity Screening

[0345] This example illustrates a method for screening concentratedculture supernatants and cell pellets from Paenibacillus strains toidentify strains possessing insecticidal activity against Coleopteranand Lepidopteran insects. DB482, which is an isolate of Paenibacillusapiarius was shown herein to contain homologues of tcaA, tcaB, tcaC, andtccC. The finding of insecticidal activity in DB482 confirms thatPaenibacillus strain DAS1529 is not unique within the genusPaenibacillus with regard to producing insecticidal activities againstLepidopteran insects. Therefore, the subject invention includes methodsused to identify other strains of Paenibacillus with insecticidalactivities against Lepidopteran insects in other species ofPaenibacillus such as P. chondroitinus, P. alginolyticus, P. larvae, P.validus, P. gordonae, P. alvei, P. lentimorbus, P. popilliae, P.thiaminolyticus, P. curdlanolyticus, P. kobensis, P. glucanolyticus, P.lautus, P. chibensis, P. macquariensis, P. azotofixans, P. peoriae, P.polymyxa, P. illinoisensis, P. amylolyticus, P. pabuli, P. macerans.

1 49 1 33521 DNA Artificial Sequence Nucleic acid sequence of the entireinsert of SB12. 1 gatcacacgg ccggcgtatt ccggctcgga accgaagaat taacagaagcgcttcagcag 60 tccggttatc ggacagtctt tgatattgca tctgaaaatc ttgcggaatttcagaaaagc 120 aatccggaga ttccctcttc cgacgcgaag gagattcatc aattagccgtccagaggaca 180 gaaaacttat gcatgcttta taaggcctgg caactgcaca atgatccggtcgtccagagc 240 cttcccaaat tatccgcgga taccggcctg cgaggcatgc gtgccgcgttggagcggagt 300 cttggagggg gagccgattt tggagacttg ttcccggagc gatcgccagagggctatgcg 360 gaagcctcct ctatacagtc gcttttttcg ccgggacgtt accttacggtgctgtataaa 420 attgcgcagg atctccacga cccaaaagac aaactgcata ttgacaaccgccgtccagat 480 ttgaagtcgc tgatcctcaa taatgacaat atgaaccgtg aggtgtcttccctggatatc 540 ctgctggatg tgctgcagtc cgaaggctcc ggcacactga catccctgaaggatacctac 600 tatccgatga cccttcccta tgatgacgac cttgcgcaaa tcaatgccgtggcggaggcg 660 cgctcatcca atttgctggg gatctgggat accctgctgg acacgcagcggacttccatc 720 ctgcaggatt ccgccgctgt ccaccggata agcaagccgc ggcactcggcatacgtcaat 780 cagagagtct ccgatgatga accggtattg atcgcgggag aggaattctacttggagacc 840 ggcggtgttg ccgacacgac cccgtctccg ccaacgaggg aagcgctttccttgacgcca 900 aacagcttcc gtctgctggt caaccccgag ccgacagcag acgacatcgccaatcactac 960 aacgttaaga ctcaagatcc tgccgctctg gccgccgtct taaatgtggtcgatgacttt 1020 tgcctgaaaa ccggtttgag ctttaatgag ttgctggact taacgatgcagaaggatgat 1080 gaatcgatcg gcagcgagta caaaagccgg tttgtaaaat ttggcggcgaggccaatgtt 1140 ccggtttcaa cctatggagc tgtatttctg acaggaacgg aagaaactccgttgtgggta 1200 ggaaaaggag ctgtgataag ccctgcagcg gacgcctatg ttcgtaatgggacatatgca 1260 aacacgaatt atggatcaga cactagtctt gttgtgaagc aggatgggtctagtggatac 1320 agtagggaag catatatcag gtttgatttg acaggtcttt ccggagttgtggaggaagct 1380 aaaatttctc taacaactag agcgaaacaa ttgtctagct taagacaccaagctcatttg 1440 gtcagtgaca acagttggga tgaattgaaa atcacatgga ataacaaacctgcaggagga 1500 gcgatcatcg caagctggga tgttcccgaa gttggtgaga atgtaaaggttgatgtgacc 1560 cggcaagtaa atgatgcgct cgcaaacggt caagataaac tatcaattgttattcgttct 1620 agtgcaaatt atggcagtct gggcgatgtc tcttatgcct ctagagaacaccctgaaaaa 1680 gcctcacgac cttctatgga aatcaaggcg ataacgggtg ctggtttaaattttacggcg 1740 gataatgttg tagctctggc aggaagggcg gaaaagcttg tccggctggcgcgcagcacg 1800 ggactttcct ttgagcagtt ggattggctg attaccaata ccagccgtgccgtaatcgaa 1860 catggtggag aactgattct ggataagccg gtactggagt ctgtggccgaattcacaagg 1920 ctccataagc gttatggcat cacagcggat atgttcgccg cgtttatcggcgaagtcaat 1980 acgtatgctg aagcaggtaa agagagcttt tatcagacga ttttcagcacggccgaccat 2040 tcggctgcct tacctttagg cgcaactttg caatttgagg tgagcaaacaggatcgatat 2100 gaagcgattt gctgcggggc catgggggtg accgccgatg agttctctcgtatcggcaaa 2160 tactgctttg gcgacaacgc gcagcaagtt accgccaatg aaacaaccgttgcgcagctt 2220 tatcgtttag gccgaattcc tcacatgctt ggattgcgtt ttaccgaggcggagctgttg 2280 tggaaattga tggctggcgg cgaggatacc ttgctccgca cgattggcgcgaagcctcgc 2340 agtttacaag ccttagagat tattcgccgt actgaggtcc ttttggactggatggatgct 2400 catcagcttg atgttgtctc cctgcaagcc atggttacca atcggtacagcggcacagcc 2460 acgccggagc tgtacaactt tttggcacag gtgcaccaat ccacaagcagtgccgcgaac 2520 gtgtccaaag cggatgctca ggataccctg cccgcggaca agctgttccgggccttggcg 2580 gtaggcttca acctgaaggc caacgtgatg gcgcaggtca ttgactggttggacaaaacc 2640 gacggagcgt ttacgctgcg ggctttctgg gacaagcttc aagcgtatttcagcgccgat 2700 catgaagaag aactgacggc cttggaagga gaagccgact tgctgcagtggtgccaacag 2760 atcagccagt atgcgctcat tgtccgctgg tgcgggttaa gcgatcaggatctggcgctg 2820 ctgaccgggc atcccgggca gcttctgtcc ggacaacata cggtgccggtaccctcgctg 2880 catctcctgc tggtgctgac ccgcctgaag gaatggcagc agcgcgtccaggtttccagc 2940 gaggaggcca tgcgctattt tgcccaggcc gatgcgccaa ccgtcacacgcgatgctgcg 3000 gtcaagctgc ttgcccgtat ccatggctgg aatgaacagg ataccgcctcgatgaatgac 3060 tacctgctgg gagagaacga atatcctaag aactttgagc agatctttactttggaaagc 3120 tgggtcaacc tgggccgtca actgaatgtt ggcagccgaa cgttgggagagctggttgac 3180 atgtcagaag aggatgatac cgcggaaaac acggatttga ttatctcggtcgcccaaagc 3240 ctgatggctg cggtgcaggc ctgaaccaac atgaccaagg aaggtggtaagaatatgtct 3300 acttcaaccc tgttgcaatt gattaaggaa tcccgccggg atgcgttggtcaaccattat 3360 atcgccaaca atgtcccgag agagcttacg gataagatta cagacgcagacagcctgtat 3420 gagtatttgc tgctggatac caagatcagt gaactcgtaa aaacatcgccgatagctgag 3480 gccattagca gcgttcagtt atacatgaac cgatgcgtgg aaggctatgaaggcaagctg 3540 actccggaag gcaacagcca tttcgggccg ggaaaattcc tgaataattgggatacctat 3600 aacaagcgtt attccacttg ggccggcaag gaacgtctga aatattatgcaggcagttat 3660 attgacccgt ccttgcgcta taacaaaacg gatccgttcc tgaacctggaacagaatatc 3720 agccagggaa gaatcaccga tgacaccgta aagaacgcgc tgcaacactacctgactgaa 3780 tatgaagtgt tggcggattt ggaatatatc agcgtaaata aaggcgccgatgaaagtgta 3840 ttattcttcg taggccgcac caaaacaatg ccatacgaat attactggcgccgattaacg 3900 ttgaaaaagg acaataacaa taaactggtg cctgccatct ggtctcaatggaaaaaaata 3960 actgccaata tcggcgaagc agttaataat tatgtggtgc ttcactggcataataaccgc 4020 ttacatgtac aatggggttc tacagagaaa acacaaaatg atgacggagaacccattgag 4080 aaacgatatt tgaatgactg gttcatggat aagtccagtg tctggtcttcattccgaaag 4140 gtttcatata tagaaaatag ttttacttat actgagggca tcattgattcaagaaatatt 4200 actatagctg gaaatcaact gttctgtgat gattcaaata cttttaaggcaacaataacg 4260 gcacttccat ttgaccaaat acgtgtttac ttagaaaaga tttacggtacaggcggcagc 4320 atcacggtta ctggagaaaa taaaggctat attattaagg tgggggagccaagagaagtc 4380 agtttctctc ctaatacgtt actagatgta ttcataggta gtaatgcaagccctcgagac 4440 ccatatttca aagctacatt taatagagaa gctctccaaa attcatacggctcaattaaa 4500 ataaatcaat acacccctcc ttctggaagc aatatcaaag gtcctatcgaccttaccctg 4560 aaaaataaca tcgacctgtc ggcgttgttg gaagagagcc ttgacgtactgttcgactat 4620 accattcagg ggaataacca attgggcggc ttagaggcct ttaacgggccttacggactt 4680 tatttgtggg aaatcttcct ccatgttcca tttttaatgg cggttcgcttccacaccgag 4740 cactgagaga tcccctcata atttccccaa agcgtaacca tgtgtgaataaattttgagc 4800 tagtagggtt gcagccacga gtaagtcttc ccttgttatt gtgtagccagaatgccgcaa 4860 aacttccatg cctaagcgaa ctgttgagag tacgtttcga tttctgactgtgttagcctg 4920 gaagtgcttg tcccaacctt gtttctgagc atgaacgccc gcaagccaacatgttagttg 4980 aagcatcagg gcgattagca gcatgatatc aaaacgctct gagctgctcgttcggctatg 5040 gcgtaggcct agtccgtagg caggactttt caagtctcgg aaggtttcttcaatctgcat 5100 tcgcttcgaa tagatattaa caagttgttt gggtgttcga atttcaacaggtaagttagt 5160 tgctagaatc catggctcct ttgccgacgc tgagtagatt ttaggtgacgggtggtgaca 5220 atgagtccgt gtcgagcgct gattttttcg gcctttagag cgagatttatacaatagaat 5280 ttggcatgag attggattgc ttttagtcag cctcttatag cctaaagtctttgagtgact 5340 agatgacata tcatgtaagt tgctgatagg tttccagttt tccgctcctaggtctgcata 5400 ttgtactttt cctcttactc gacttaacca gtaccaaccc agcttctcaacggatttata 5460 ccatggcact ttaaagccag catcactgac aatgagcggt gtggtgttactcggtagaat 5520 gctcgcaagg tcggctagaa attggtcatg agctttcttt gaacattgctctgaaagcgg 5580 gaacgctttc tcataaagag taacagaacg accgtgtagt gcgactgaagctcgcaatac 5640 cataagccgt ttttgctcac ggatatcaga ccagtcaaca agtacaatgggcatcgtatt 5700 gcccgaacag ataaagctag catgccaacg gtatacagcg agtcgctctttgtggaggtg 5760 acgattacct aacaatcggt cgattcgttt gatgttatgt tttgttctcgctttggttgg 5820 caggttacgg ccaagttcgg taagagtgag agttttacag tcaagtaaggcgtggcaagc 5880 caacgttaag ctgttgagtc gttttaagtg taattcgggg cagaattggtaaagagagtc 5940 gtgtaaaata tcgagttcgc acattttgtt gtctgattat tgatttttggcgaaaccatt 6000 tgatcatatg acaagatgtg tatctacctt aacttaatga ttttgataaaaatcattagg 6060 ggattcatca gcaccgagca gcggtatgag ttggcggaac gatggtttaaattcattttc 6120 aacagcgcag gttaccgtga tggctacggc aatctgctga cggatgacaaaggcaacgtg 6180 cgctactgga acgtcgtgcc tctgcaggag gatacggagt gggatgacacgttgtccctg 6240 gcaacgaccg acccggacga gattgcgatg gccgacccga tgcaatacaagctggctatc 6300 tttattcaca ccttggactt cttgatcagc cgcggcgaca gcttgtaccggatgctggag 6360 cgggatacct tgaccgaagc gaagatgtat tacattcagg ccagccaactgcttgggcct 6420 cgtcccgaga tccggatcaa tcacagctgg cctgatccga ccctgcaaagcgaagcggac 6480 gcggtaaccg ccgtgccgac gcgaagcgat tcgccggcag cgccaattctcgccttgcga 6540 gcgcttctga atgcggaaaa cgggcatttc ctgccgcctt ataatgatgaactattagct 6600 ttctgggata aaatcgacct gcgtctctac aatttacgcc acaatctgagcctggacggt 6660 cagccgcttc atttgccgct ctttaccgaa ccggtcaatc ctcgtgagctgcaggttcag 6720 catggggcag gcgatggatt agggggaagc gccggttccg tccaaagccgtcaaagtgtc 6780 tatcgttttc ctctggtcat cgataaggcg cgcaatgccg cgagtagtgttatccaattc 6840 gggaatgccc tggaaaacgc gctgacaaag caggacagcg aggccatgactatgctgttg 6900 caatcccagc agcagattgt cctgcagcaa acccgcgata ttcaggagaagaacctggcc 6960 tcgctgcaag caagtctgga agcaacgatg acagccaaag cgggcgcgaaatcccgaaag 7020 acccattttg ccggcctggc ggataactgg atgtcgcata atgaaaccgcctcacttgca 7080 ctgcgtacca ctgcgggaat tatcaataca agctcgaccg tgccaatcgctatcactggc 7140 ggcttggata tggctccgaa catttttggt ttcgcagttg gaggttcccgctggggagca 7200 gccagcgcgg ctgtagccca aggattgcaa atcgccgccg gcgtaatggaacagacggcc 7260 aatatcatcg atatcagcga aagctaccgc cggcgccggg aggattggctgctgcagcgg 7320 gatgttgccg agaatgaagc ggcgcagttg gattcgcaga ttgcggccctgcgggaacag 7380 atggatatgg cgcgaaaaca acttgcgctg gcggagacgg aacaggcacacgcgcaagcg 7440 gtctacgagc tgctaagcac ccgttttacg aatcaagctt tgtataactggatggccgga 7500 cgtctgtcgt ctctatacta tcaaatgtat gacgccgcat tgccgctctgcttgatggcc 7560 aaacaggctt tagagaaaga aatcggcaat gataaaacgg ttggaatcttcaccctcccg 7620 gcctggaatg atttgtatca gggattgcta gcgggcgagg cgctgctgctcgagcttcag 7680 aagctggaga atctgtggct ggaggaggac aagcgcggaa tggaagctgtaagaacggta 7740 tctttagata cccttctccg caaagaaaag ccagaatccg gttttgcagatttcgtcaag 7800 gaagttctgg acggaaagac gcctgaccct gtaagcggag ttagcgtacagctgcaaaac 7860 aatattttca gtgcaaccct tgacctgtcc acccttggcc tggatcgcttttacaaccaa 7920 gcggaaaagg cccacaggat caaaaacctg tcggttacct tacccgcgctattgggacct 7980 tatcaggata ttgcggcaac cttatcgcta ggtggcgaga ccgttgcgctttcccatggc 8040 gtggatgaca gcggcttgtt tatcacggat ctcaacgaca gccgtttcctgcctttcgag 8100 ggtatggatc ctttatccgg cacactcgtt ctgtcgatac tccatgccgggcaagacggt 8160 gaccagcgcc tcctgctgga aagcctgaac gacgtcatct tccacattcgatatgtcatg 8220 aaatagaaga caaactcccg cgaaatagtt caaccgcggg agttctttattttccaccca 8280 aatcattgac ataaatatac tttaataata tgttggagga agaagaggaggttgttattg 8340 gtgagaataa agaaaatgtt tgaagtagcg atgatgttat cattggcgtgtttgtttttt 8400 gttacatcag ctgcttcagc aaaaacaact aatttaactt cttccccgaaacttatgaac 8460 tgctttgatg tagctggtaa cgtaacctac aaaaccgctc ctgatggttcaattacgaaa 8520 ataatcgaag tacaagatgt tagtaaattc agtgaacaaa ccaatctcaggttggctcca 8580 gattctaaag ttaccattta tattcctgac tccagtgaca ataaccagcccaattactcc 8640 aataataatt ccaatgacta tagtgaacaa aattacctca atactaaccccaacgttgaa 8700 ccatttgttg aaccatttgt tgaacttata aagtatattg cgaacgtaagtgatccatat 8760 gaagcgtgtg gatcaaaatc gattagagat tctgattatg atcctcccggcggaaaaatg 8820 ataataaaac aagggataca ggctacacac tcaaccacgg tttctatcgatgccaaaatc 8880 gtttcaactg ccttaaaata tgatgtaaca acgagttatt ccattgaagaggagcaaaat 8940 attaaagtac cagacaataa aagaggaaga attattgctt atccaaagtatgatgtcaac 9000 acctttgaaa tatgggaagc tggtctaata tataataaaa agattggagacggtacggct 9060 ttctatccta aaggagtatg ttttgttaca attattaatt aactttcaaataagagaagc 9120 tgtttcttaa gaataaagaa gcagcttctt gcatttttta ttatgatattacactctatc 9180 ttctgtcaga tgctccctct caacttccat tcccaatccc ctcttcttaaaaggccataa 9240 aagttacact tatcatttcc gtcattgcta atctaccttg cagttaacctaaaatatacc 9300 ttccgggatt cctgaaggat gaaacatatt ttcacccatc agtgaaactatctatgcttt 9360 tttgattgaa gcgagaggta tgcttgggtt gtaaatgaaa ggggggacctgttcatggga 9420 aactacctaa aacggtaacg gatagcttct atggttttga tgtctgcagtcattttgtct 9480 tggggagtac tcatcattca gaacacaaga ggaggagttc atggtgtcaacaacagacaa 9540 cacggccggc gtattccggc tcggaaccga agaattaaca gaagcgcttaagcagtccgg 9600 ttatcggacc gtctttgata ttgtatctga caatcttgcg gaatttcagaaaaacaatcc 9660 ggagattccc tcttctgacg cgaaggagat tcatcaatta gccgtccagaggacagaaaa 9720 cttatgcatg ctttataagg cctggcagct gcacaatgat ccggttgtccagagccttcc 9780 caaattatcc gcggataccg gcctgcaagg catgcgtgcc gcgttggagcggagtcttgg 9840 aggcggagcc gattttggag acttgttccc ggagcgatcg ccagagggctatgcggaagc 9900 ctcctctata cagtcgcttt tctcgccggg acgttacctg acggtgctgtataaaattgc 9960 gcgggatctc cacgacccaa aagataaact gcatattgac aaccgccgtccagatttgaa 10020 gtcgctgatc ctcaataatg acaatatgaa ccgagaggta tcttctctggatatccttct 10080 ggatgtgctg cagcccgaag gctctgacac gctgacatcc ttgaaggatacctaccatcc 10140 gatgaccctt ccctatgatg acgaccttgc gcaaatcaat gccgtggcggaggcgcgttc 10200 atctaatttg ctggggattt gggataccct gctggacacg cagcggacttccatcctgca 10260 gaattccgcc gctgcccgcc ggataagcaa ggcgcggcac tcggcatacgccaatcagaa 10320 agcctccaat gatgagccgg tattcatcac gggagaggaa atctacctggaaaccggagg 10380 taaacggctt tttctggcgc ataaactcga gataggttca actattagcgctaaaatcaa 10440 cattggaccg ccgcaagcgg ccgatatcgc gccggcaaag ttgcaactcgtatattacgg 10500 cagaggcggc agagggaact acttcctgcg cgtggcagac gatgtgtccctcggtggaaa 10560 gctgctgacc aattgttatc tgaccagcga tgacggacag agcaacaatattagcgggcc 10620 atactgccta atgatcaacc gaggcaccgg cagcatgcct agcgggactcaccttccagt 10680 tcagattgaa agagtgaccg atacatccat ccgcattttt gtgccggatcacggctattt 10740 ggggctaggc gaaagccttg ccagcaactg gaatgaaccg ttggcgctgaatctgggctt 10800 ggatgaagcg ttgaccttta ccttgagaaa gaaggagacg ggaaatgacaccatttccat 10860 aatcgacatg ctgccgccgg tagcgaacac gactccgtct ccgccgacgagggaaacgct 10920 ttccttgacg ccaaacagct tccgtctgct ggtcaaccct gagccgacagcggaggacat 10980 cgccaagcac tacaacgtca cgacggtaac ccgggctcct gccgatctggcctccgcctt 11040 aaatgttgtc gatgatttct gcttgaaaac cggtttgagc tttaacgaattgctggattt 11100 aaccatgcag aaggattatc agtcaaaaag cagtgagtac aaaagccgatttgtaaaatt 11160 cggcggcggg gagaatgttc cggtatcaag ctatggcgca gcctttctgacaggagcgga 11220 agatactcct ttgtgggtga aacagtataa cagcgtgggg actgcaacaagcacccctgt 11280 tttaaacttt acgccagata atgttgtggc tttggcagga agggcggaaaagcttgtccg 11340 gctgatgcgc agcacgggtc tttcctttga gcagttggat tggctgattgccaatgccag 11400 ccgtgccgtt atcgaacacg gtggagagct ttttctggat aagccggtactggaagctgt 11460 ggccgaattc acaaggctca ataagcgtta tggcgtcaca tcggatatgttcgccgcgtt 11520 tatcggcgaa gtcaatacgt atacagaagc gggcaaggac agcttttatcaggcgagttt 11580 cagcacggcc gaccattcgg ctaccttacc tttgggcgct tctttgcaacttgaggtgag 11640 caagcaggat cgatatgaag cgatttgctg cggggctatg ggggtgaccgccgatgagtt 11700 ctcccgtatc ggcaaatact gctttgggga taaagcacag caaatcacggccaatgaaac 11760 aaccgttgcc cagctttatc gtttaggccg aattcctcat atgctaggcttgcgttttac 11820 cgaggcagag ctgttgtgga aattgatggc tgggggcgag gataccttgctccgcacgat 11880 tggcgcgaac cctcgcagtt tagaagcgtt agagattatt cgccggacggaggtcctttt 11940 ggactggatg gatgcccatc agctggatgt tgtctccctg caagccatggttaccaatcg 12000 gtacagcggc acagccacgc cggagctgta caattttttg gcacaggtgcatcaatccgc 12060 aagcagtgcc gcgaacgtgg ccagagcgga tggtcaggat acgttgcctgcggacaagct 12120 gctccgggca ttggcggcgg gcttcaaact gaaagccaac gtgatggcgcgagtaatcga 12180 ctggatggac aaaaccaata aagcgtttac gctgcgggct ttctgggacaagcttcaagc 12240 gtatttcagc gccgatcatg aagaagaact gaccgccctg gaaggagaagccgcaatgct 12300 gcagtggtgc cagcagatca gccagtatgc gctcattgtc cgctggtgcgggttaagcga 12360 gcaggatctg gcgctgctga ccgggaatcc ggagcagctt ctggacggacaacatacggt 12420 gcccgtaccc tcgctgcatc tcctgctggt gctgacccgc ctgaaggaatggcagcagcg 12480 cgtccaggtt tccagcgagg aggctatgcg ctattttgcc caggccgattcgccaaccgt 12540 cacgcgcgac gatgcggtta atctgcttgc ccgtatccat ggctggaatgaagcggatac 12600 cgtctcgatg aatgactacc tgctgggaga gaacgaatat cctaagaactttgatcagat 12660 ctttgcactg gaaagctggg tcaacctggg ccgtcaactg aacgtgggcagcagaacgct 12720 gggagagctg gttgacatgg ctgaagagga taaaaccgcg gaaaacatggatctgattac 12780 ttcggtggcc catagcctga tggctgcagc gaaagcctga accaacatgaccaaggaagg 12840 tgataagcat atgtctactt caaccctgtt gcaatcgatt aaagaagcccgccgggatgc 12900 gctggtcaac cattatattg ctaatcaggt tccgacagcg cttgcggacaagattacgga 12960 cgcggacagc ctgtatgagt acttgctgct ggataccaag atcagtgaactcgtaaaaac 13020 atcgccgata gcggaggcca tcagcagcgt gcagttatac atgaaccgctgcgtcgaagg 13080 ctatgaaggc aagttgactc cggaaagtaa tactcatttt ggcccaggtaaatttctata 13140 taactgggat acgtacaaca aacgtttttc cacctgggca ggaaaagaacgcttgaaata 13200 ttatgcaggc agctatattg agccgtcctt gcgctacaac aaaaccgatccattcctgaa 13260 cctggaacag agcatcagcc agggaagaat tactgatgat accgtaaagaacgcgctgca 13320 acactacctg actgaatatg aagtgttggc ggatctggat tatatcagcgttaataaagg 13380 cggcgacgaa agtgttttac tctttgttgg acgcaccaaa accgtaccgtatgaatacta 13440 ctggcgccgt ttgcttttaa aaagggacaa taataataag ctagtaccagcagtctggtc 13500 tcagtggaaa aaaatcagtg ccaatatcgg tgaagcggtt gatagttatgtggtgcctcg 13560 gtggcataaa aaccggctac atgtgcaatg gtgttctata gagaaaagtgaaaatgatgc 13620 cggtgaaccc attgagaaac gatatttgaa tgactggttc atggatagttccggagtctg 13680 gtcttcattt cgaaagattc cggttgtgga aaagagtttc gaatatttggacggaagcct 13740 cgatccccga tttgtcgctc ttgttagaaa tcaaatatta attgatgagccagaaatatt 13800 cagaattaca gtatcagccc ctaatccgat agatgcaaat ggaagagtagaggtacattt 13860 tgaagaaaac tatgcaaaca gatataatat taccattaaa tatgggacaacgagtcttgc 13920 tattcctgca gggcaggtag ggcatccaaa tatctctatt aatgaaacattaagggttga 13980 attcggcacc aggccggatt ggtattatac tttcagatat ttaggaaatacaatccaaaa 14040 ctcatacggt tcaattgtca ataatcaatt ttcacctcca tcaggaagcaatattaaagg 14100 tcctatcgac cttaccctga aaaataacat cgacctgtcg gccttgttggatgagagcct 14160 tgacgcactg ttcgactata ccattcaggg cgataaccaa ttgggcggcttagctgcctt 14220 taacgggcct tacggacttt acttgtggga aatcttcttc catgttccttttttaatggc 14280 ggttcgcttc cacaccgagc agcggtatga gttggcggaa cgttggtttaaattcatctt 14340 caacagcgca ggataccgtg atgattacgg cagtctgctg acggatgacaaaggcaacgt 14400 gcgttactgg aacgtgatac cgctgcaaga ggacacggag tgggatgacacgttgtccct 14460 ggcaacgacc gacccggacg agattgcgat ggccgacccg atgcaatacaagctggctat 14520 atttattcac accatggact tcctgatcag ccgcggcgat agcttgtaccggatgctgga 14580 gcgggatacc ctggccgaag ccaagatgta ttacattcag gccagccaactgcttgggcc 14640 ccgccccgac atccggctca atcacagttg gcctaatccg accttgcaaagcgaagcgga 14700 cgcggtaacc gccgtgccga cgcgaagcga ttcgccggca gcgccaattttggccttgcg 14760 agcgcttctg acaggcgaaa acggtcattt cctgccgcct tataatgatgaactgttcgc 14820 tttctgggac aaaatcgatc tgcgtttata caatttgcgc cacaatttgagtctggacgg 14880 tcagccgctt catttgccgc tctttgccga accggtcaat ccgcgtgaattgcaggttca 14940 gcatggcccg ggcgatggct tggggggaag cgcgggttcc gcccaaagccgtcagagtgt 15000 ctatcgtttt cctctggtca tcgataaggc gcgcaatgcg gccaacagtgtcatccaatt 15060 cggcaatgcc ctggaaaacg cactgaccaa gcaagacagc gaagcaatgaccatgctgtt 15120 gcagtcccag cagcagattg tcctgcagca aacccgcgat attcaggagaagaacctggc 15180 cgcgctgcaa gcaagtctgg aagcaacgat gacagcgaaa gcgggggcggagtcccggaa 15240 gacccatttt gccggcttgg cggacaactg gatgtcggac aatgaaaccgcctcactcgc 15300 actgcgtacc accgcgggaa tcatcaatac cagctcaacc gtgccgatcgccatcaccgg 15360 cggcttggat atggctccga acatttttgg tttcgcagtt ggaggttcccgctggggagc 15420 agccagcgcg gctgtagccc aaggattgca aatcgccgcc ggcgtaatggaacagacggc 15480 caatattatc gatattagcg aaagctaccg ccggcgccgg gaggattggctgctgcagcg 15540 ggatgttgcc gaaaatgaag cggcgcagtt ggattcgcag attgcggccctgcgggaaca 15600 gatggatatg gcgcgcaagc aacttgcgct ggcggagacg gaacaggcgcacgcgcaagc 15660 ggtctacgag ctgcaaagca cccgctttac gaatcaagct ttgtataactggatggctgg 15720 acgtctgtcg tctctatact atcaaatgta tgacgccgca ttgccgctctgcttgatggc 15780 gaagcaggct ttagagaaag aaatcggttc ggataaaacg gtcggagtcttgtccctccc 15840 ggcctggaat gatctatatc agggattatt ggcgggcgag gcgctgctgctcgagcttca 15900 gaagctggag aatctgtggc tggaggaaga caagcgcgga atggaagccgtaaaaacagt 15960 ctctctggat actcttctcc gcaaaacaaa tccgaactcc gggtttgcggatctcgtcaa 16020 ggaggcactg gacgaaaacg gaaagacgcc tgacccggtg agcggagtcggcgtacagct 16080 gcaaaacaat attttcagcg caacccttga cctctccgtt cttggcctggatcgctctta 16140 caatcaggcg gaaaagtccc gcaggatcaa aaatatgtcg gttaccttacctgcgctatt 16200 ggggccttac caggatatag aggcaacctt atcgctaggc ggcgagaccgttgcgctgtc 16260 ccatggcgtg gatgacagcg gcttgttcat cactgatctc aacgacagccggttcctgcc 16320 tttcgagggc atggatccgt tatccggcac actcgtcctg tcgatattccatgccgggca 16380 agacggcgac cagcgcctcc tgctggaaag tctcaatgac gtcatcttccacattcgata 16440 tgttatgaaa tagctttaca gtcagatata ttccggggct tgtattcacaagcccctcca 16500 aggaggaatt gggttatgcc acaatctagc aatgccgata tcaagctattgtcgccatcg 16560 ctgccaaagg gcggcggttc catgaaggga atcgaagaaa acatcgcggctcccggctcc 16620 gacggcatgg cacgttgtaa tgtgccgctg ccggtaacct ccggccgctatattactcct 16680 gatataagcc tgtcctatgc gagcggccac ggcaacggcg cttatggaatgggctggacg 16740 atgggagtga tgagcattag ccggagaaca agccgaggga cccccagttatacatccgaa 16800 gaccagttcc ttggtccgga tggggaggtg cttgttccgg aaagcaacgaacaaggggag 16860 atcattaccc gccacaccga tacggcccaa gggataccgt taggcgagacgtttacggtt 16920 acacgctatt ttccccggat cgagagcgct tttcatttgc tggaatactgggaagcgcaa 16980 gcaggaagcg caacagcgtc gttttggctt attcactctg ccgatggagtgctgcactgt 17040 ctgggtaaaa ctgctcaggc gaggatagcc gcccctgacg attccgccaagatcgcagaa 17100 tggctagtgg aggagtccgt ctcccccttc ggagagcata tttattaccaatacaaagaa 17160 gaagacaatc aaggcgtgaa tctggaggaa gacaatcatc aatatggggcgaaccgctat 17220 ctgaaatcga ttcgctatgg aaataaggtt gcctctcctt ctctctatgtctggaagggg 17280 gaaattccgg cagacggcca atggctgtat tccgttatcc tggattatggcgagaacgat 17340 acctcagcgg atgttcctcc cctatacacg ccccaagggg agtggctggtgcgcccggac 17400 cgtttttccc gctatgacta cggatttgag gtccggactt gccgcttgtgccgccaggtc 17460 ttgatgttcc acgtctttaa ggagcttggc ggggagccgg cgctggtgtggcggatgcag 17520 ttggaatacg acgagaaccc ggcggcgtcc atgctgagcg cggtccggcaattggcttat 17580 gaagcagatg gggccattcg aagcttgccg ccgctggaat tcgattatactccatttggc 17640 atcgagacaa cggccgattg gcagcctttt ctgcctgtgc ctgaatgggcggatgaagaa 17700 cattatcagt tggtcgattt gtacggagaa ggcataccgg gcttattatatcagaacaat 17760 gaccactggc attatcgttc gcccgcccgg ggcgacacac cggacgggatcgcctataac 17820 agctggcggc cgcttcctca tatccccgtg aactcccgga acgggatgctgatggatctg 17880 aatggagacg ggtatctgga atggttgctt gcggaacccg gggttgcggggcgctatagc 17940 atgaacccgg ataagagctg gtccggtttt gtgccgctcc aggcactgccaacggaattc 18000 ttccatccgc aggcacagct tgccaatgtt accggatcgg gtttaaccgacttggttatg 18060 atcggtccga agagcgtccg gttttatgcc ggagaagaag cgggcttcaagcgcgcatgt 18120 gaagtgtggc agcaagtggg cattactttg cctgtggaac gcgtggataaaaaggaactg 18180 gtggcattca gcgatatgct gggatcgggt cagtctcatc tggtgcgcatccggcatgat 18240 ggcgttacat gctggcctaa tctggggaac ggcgtgttcg gggcgccgttggcccttcac 18300 gggtttacgg catcggagcg ggaattcaat ccggaacgtg tatatcttgtggaccttgat 18360 ggatccggcg cttccgatat catttatgct tctcgtgacg ctctactcatttaccgaaat 18420 ctttccggca atggctttgc tgatccggtg cgggttccgc tgcctgacggcgtgcggttt 18480 gataatctgt gccggctgct gcctgccgat atccgcgggt taggtgtggccagtctggtg 18540 ctgcatgtac cttacatggc cccccgcagt tggaaattag atttctttgcggcgaagccg 18600 tatttattgc aaacggtcag caacaatctt ggagcttcca gctcgttttggtaccgaagc 18660 tccacccagt attggctgga tgagaaacag gcggcctcat cggctgtctccgctttgccc 18720 ttcccgataa acgtggtatc ggatatgcac acggtggacg aaatcagcggccgcaccagg 18780 actcagaagt atacttaccg ccatggcgtg tatgaccgga ccgaaaaggaatttgccgga 18840 ttcggccgca ttgacacatg ggaagaggag cgggattccg aaggaaccctgagcgtcagc 18900 actccgcccg tgctgacgcg gacctggtat cataccgggc aaaagcaggatgaggagcgt 18960 gccgtgcagc aatattggca aggcgaccct gcggcttttc aggttaaacccgtccggctt 19020 actcgattcg atgcggcagc ggcccaggat ctgccgctag attctaataatgggcagcaa 19080 gaatactggc tgtaccgatc attacaaggg atgccgctgc ggactgagatttttgcggga 19140 gatgttggcg ggtcgcctcc ttatcaggta gagagcttcc gttatcaagtgcgcttggtg 19200 cagagcatcg attcggaatg tgttgccttg cccatgcagt tggagcagcttacgtacaac 19260 tatgagcaaa tcgcctctga tccgcagtgt tcacagcaga tacagcaatggttcgacgaa 19320 tacggcgtgg cggcacagag tgtaacaatc caatatccgc gccgggcacagccggaggac 19380 aatccgtacc ctcgcacgct gccggatacc agctggagca gcagttatgattcgcagcaa 19440 atgctgctgc ggttgaccag gcaaaggcaa aaagcgtacc accttgcagatcctgaaggc 19500 tggcgcttga atattcccca tcagacacgc ctggatgcct tcatttattctgctgacagc 19560 gtgcccgccg aaggaataag cgccgagctg ctggaggtgg acggcacgttacgatcttcg 19620 gcgctggaac aggcttatgg cggccagtca gagatcatct atgcgggcgggggcgaaccg 19680 gatttgcgag ccctggtcca ttacaccaga agcgcggttc ttgatgaagactgtttacaa 19740 gcctatgaag gcgtactgag cgatagccaa ttgaactcgc ttcttgcctcttccggctat 19800 caacgaagcg caagaatatt gggttcgggc gatgaagtgg atatttttgtcgcggaacaa 19860 ggatttaccc gttatgcgga tgaaccgaat tttttccgta ttctggggcaacaatcctct 19920 ctcttgtccg gggaacaagt attaacatgg gatgataatt tctgtgcggttacatccatc 19980 gaagacgcgc ttggcaatca aattcagatt gcatatgatt accgctttgtggaggccatc 20040 cagattaccg atacgaataa taatgtgaat caggtcgccc tggatgctctcggccgggtc 20100 gtatacagcc ggacctgggg cacggaggaa gggataaaga ccggcttccgcccggaggtg 20160 gaattcgcga cgcccgagac aatggagcag gcgcttgccc tggcatctcccttgccggtt 20220 gcatcctgct gtgtatatga tgcgcatagc tggatgggaa cgataactcttgcacaactg 20280 tcagagcttg ttccagatag tgaaaagcaa tggtcgttct tgatagacaatcgcttgatt 20340 atgccggacg gcagaatcag atcccgcggt cgggatccat ggtcgcttcaccggctattg 20400 ccgcctgctg tgggcgaatt gctgagcgag gcggaccgta aaccgccgcatacggtaatt 20460 ttggcagcag atcgttaccc ggatgaccca tcccagcaaa ttcaggcgagcatcgtgttt 20520 agcgatggct ttgggcgtac gatacaaact gctaaaagag aagatacccgatgggcgatt 20580 gcggaacggg tggactatga cggaaccgga gccgtaatcc gcagctttcagcctttttat 20640 cttgacgact ggaattatgt gggcgaagag gctgtcagca gctctatgtacgcaacgatc 20700 tattattatg atgctctggc acgacaatta aggatggtca acgctaaaggatatgagagg 20760 agaactgctt tttacccatg gtttacagta aacgaagatg aaaatgataccatggactca 20820 tcattatttg cttcaccgcc tgcgcggtga gatggaggtt aaacctatgaacacaacgtc 20880 catatatagg ggcacgccta cgatttcagt tgtggataac cggaacttggagattcgcat 20940 tcttcagtat aaccgtatcg cggctgaaga tccggcagat gagtgtatcctgcggaacac 21000 gtatacgccg ttaagctatc ttggcagcag catggatccc cgtttgttctcgcaatatca 21060 ggatgatcgc ggaacaccgc cgaatatacg aaccatggct tccctgagaggcgaagcgct 21120 gtgttcggaa agtgtggatg ccggccgcaa ggcggagctt tttgatatcgaggggcggcc 21180 cgtctggctt atcgatgcca acggcacaga gacgactctc gaatatgatgtcttaggcag 21240 gccaacagcc gtattcgagc aacaggaagg tacggactcc ccccagtgcagggagcggtt 21300 tatttatggt gagaaggagg cggatgccca ggccaacaat ttgcgcggacaactggttcg 21360 ccactacgat accgcgggcc ggatacagac cgacagcatc tccttggctggactgccgtt 21420 gcgccaaagc cgtcaactgc tgaaaaattg ggatgaacct ggcgactggagtatggatga 21480 ggaaagcgcc tgggcctcgt tgctggctgc cgaagcttat gatacgagctggcggtatga 21540 cgcgcaggac agggtgctcg cccaaaccga cgccaaaggg aatctccagcaactgactta 21600 caatgacgcc ggccagccgc aggcggtcag cctcaagctg caaggccaagcggagcaacg 21660 gatttggaac cggatcgagt acaacgcggc gggtcaagtg gatctcgccgaagccgggaa 21720 tggaatcgta acggaatata cttacgagga aagcacgcag cggttaatccgaaaaaaaga 21780 ttcccgcgga ctgtcctccg gggaaagaga agtgctgcag gattatcgttatgaatatga 21840 tccggtaggc aatatccttt ctatttacaa tgaagcggag ccggttcgttatttccgcaa 21900 tcaggccgtt gctccgaaaa ggcaatatgc ctacgatgcc ttgtatcagcttgtatctag 21960 ttcggggcgg gaatccgacg cgcttcggca gcagacgtcg cttcctcccttgatcacgcc 22020 tatccctctg gacgatagcc aatacgtcaa ttacgctgaa aaatacagctatgatcaggc 22080 gggcaattta atcaagctta gccataacgg ggcaagtcaa tatacaacgaatgtgtatgt 22140 ggacaaaagc tcaaaccggg ggatttggcg gcaaggggaa gacatcccggatatcgcggc 22200 ttcctttgac agagcaggca atcaacaagc tttattcccg gggagaccgttggaatggga 22260 tacacgcaat caattaagcc gtgtccatat ggtcgtgcgc gaaggcggagacaacgactg 22320 ggaaggctat ctctatgaca gctcgggaat gcgtatcgta aaacgatctacccgcaaaac 22380 acagacaacg acgcaaacgg atacgaccct ctatttgccg ggcctggagctgcgaatccg 22440 ccagaccggg gaccgggtca cggaagcatt gcaggtcatt accgtggatgagggagcggg 22500 acaagtgagg gtgctgcact gggaggatgg aaccgagccg ggcggcatcgccaatgatca 22560 gtaccggtac agcctgaacg atcatcttac ctcctcttta ttggaagttgacgggcaagg 22620 tcagatcatt agtaaggaag aattttatcc ctatggcggc acagccctgtggacagcccg 22680 gtcagaggta gaggcaagct acaagaccat ccgctattca ggcaaagagcgggatgccac 22740 aggcctgtat tattacggac accgctacta tatgccatgg ttgggtcgctggctgaatcc 22800 ggacccggcc ggaatggtag atggactaaa cctgtaccgt atggtcaggaacaatcctat 22860 aggactgatg gatccgaatg ggaatgcgcc aatcaacgtg gcggattatagcttcgtgca 22920 tggtgattta gtttatggtc ttagtaagga aagaggaaga tatctaaagctatttaatcc 22980 aaactttaat atggaaaaat cagactctcc tgctatggtt atagatcaatataataataa 23040 tgttgcattg agtataacta accaatataa agtagaagaa ttgatgaaatttcaaaaaga 23100 cccacaaaaa gccgcacgga aaataaaggt tccagaaggg aatcgtttatcgaggaacga 23160 aaattatcct ttgtggcacg attatattaa cattggagaa gctaaagctgcatttaaggc 23220 ctctcatatt ttccaagaag tgaaggggaa ttatgggaaa gattattatcataaattatt 23280 attagacaga atgatagaat cgccgttgct gtggaaacga ggcagcaaactcgggctaga 23340 aatcgccgct accaatcaga gaacaaaaat acactttgtt cttgacaatttaaatatcga 23400 gcaggtggtt acgaaagagg gtagcggcgg tcagtcaatc acagcttcggagctccgtta 23460 tatttatcga aatcgcgaaa gattgaacgg gcgtgtcatt ttctatagaaataatgaaag 23520 gctagatcag gctccatggc aagaaaatcc ggacttatgg agcaaatatcaaccgggtct 23580 tagacaaagc agcagttcaa gagtcaaaga acgagggatt gggaactttttccgccggtt 23640 ttcaatgaag agaaagtagc atgtaactaa aattgctccc cattggttgtgtaaactaat 23700 ggggagttgt gattcactcc tgttcaacgc cattcatgta gaattgttttgggaggttaa 23760 accgattgga tgccggcccc aaggcggagc tttttgatac cgaggggcttcgagtgtggc 23820 ttatcgatat caacggcaca cagacgactc tcgaatatga tgtattaggcaggcctgcag 23880 ccgtattcga gcatcaggaa ggcaaggaat ttcctaagtg ccgggatcggtttagaatga 23940 gtctgatgcc aagccaacaa tttgcgaggg cagttgatgc gccactacgatacaatcccg 24000 ttacattcct tgataataag gggcttaaaa tcgtaattac cctaagtctcgtcgaggttg 24060 ctatagaatt gtatcgtctt catggtggcg ttcttttgct tcataatagtacgtgctgct 24120 agaattgtgc aggacgtcgc acattgctga tgtaaatgta tgtctttttcttggaatagt 24180 agatccgctc cttgttctga tgtgttcatt ctactagccc ttattttttctggccaacta 24240 agtcctatat ataattataa aaaaagcata gatatcttca tctataggtgaggatatcta 24300 tgcttttcat tttttgatta gagatatact tgtagtgcaa ggaaaagtagataggagggt 24360 gaatttaaca gaagttacaa actgttgttt acttaaaaaa ttaatatggagggaaataaa 24420 tatgaactca aatgaaccaa atttatctga tgttgttaat tgtttaagtgaccccaatag 24480 tgacttggag aagtctggcg gtggagtagc gctagatgtt ggaatgtcattgatatccga 24540 acttcttggt acggttccag ttgctggatc aattcttcaa tttgtattcgataaattgtg 24600 gtttattttt ggcccttctg agtgggactc acttatggaa catgttgaagcattaattga 24660 tagtaaaata caagagcagg taaaaagaag tgcacaagat gaactaaatgcaattacaaa 24720 taacttatct acgtatttga aatttctaga tgcatgggaa aatgattctaataatttaag 24780 agcgagagct gtagtgaaag accaatttgt aggccttgaa cagactcttgaaagaaaaat 24840 ggttagtgtt tttggaagta cgggtcatga agtgcatctt ttgccaattttcgctcaagc 24900 agccaacctc cacctaattc tattaagaga tgctgagaaa tatggaaagagatggggttg 24960 ggcagataga gaaattcaag tatattatga taaccagatt cgttatatccatgaatatac 25020 ggaccattgt attaaatatt ataatcaagg attaagtaaa ctgaaaggttctacctatca 25080 agattgggat aagtataatc gttttagaag agaaatgacc ctaactgttcttgatttgat 25140 ttcaattttc ccatcgtatg atactagaac ttacccaatt gatacaataggtcaattgac 25200 aagggaagtt tattcggatt tacttattgc taacccgtct gggatgcagactttcactaa 25260 tgtagatttc gacaatattc ttattagaaa acctcattta atggatttcttaagaactct 25320 tgagattttt accgatcgac ataacgcaag cagacacaac gtatattggggcggacatcg 25380 agtgcattct tcttacacag gaggtaattt tgaaaatttt gaatctcccttatatggcag 25440 tgaagcaaat gtagaacccc gaacatggtt gagttttgga gaatctcaagtctataatat 25500 acgttcgaag cctgagtggg atagaggaag tactgcaatt agtggctcctatgaatttcg 25560 aggagtgaca ggatgttctt tttatcgaat gggaaatttt gctggcaccgtagccctaac 25620 ttaccgacag tttggtaacg aaggttctca aatcccattg cacaggctatgtcatgttac 25680 ttattttaga agatctcaag ctgtgggggc gacttcgaga cagacgttaacaagtggtcc 25740 gctattttcc tggacacata gtagtgctac ggaaacgaat atcattcacccgacaaaaat 25800 tacacaaata ccaatggtga aggctagttc ccttggatca ggtacttctgttgtccaagg 25860 accaggcttt acaggagggg atgtacttcg aagaaatagc cccggtagcacaggaacttt 25920 aagagttaac gtcaattcac cattatcaca gagatatcgt ataagaattcgttacgcttc 25980 tactacggat ttagattttt ttgtcattcg cggaaatacg acagttaataattttagatt 26040 tgggaacact atgcgtaaag gagaccctat aacctctcga tcatttagatttgcggcttt 26100 tagtacacca tttacttttg ctagctcaca ggatgaactt agaataaatgtacaaaattt 26160 caataatggt gaagaagttt atatagatag aatcgaagtt attccagtttgatactacag 26220 atgtttatgt tgatacccta ggtaatacac ctactgagac gttgggaagaagtgtaaaaa 26280 actcatctca aaatcgaaag taaaagagcc cttcttaaaa ttttaagaggggctcagctc 26340 attgttgcac agttactcca gggtctgtca aggattttgc ttaaattgatttatatgaac 26400 ctaccccgag ggggagacga gcctcaaggc aaaatgctgg ccgatgcgttctggaaagaa 26460 tacggagaac tgcagcagca ttttcggaca gcaggcggaa atagcctgagagaagacgat 26520 gaggttgtcg acccaggtga tcaggatgtc ctggactccc cgattcttaagatcgttcag 26580 aatgctgagc cagaacttcg tggactcgtt ttccccgatc cacatgctcagtacgacctt 26640 attgccgtcc aaatcgatgc cgtacacatt ctggagatga tcttcaatctcccgtgtact 26700 tacgtcctct tggcgtagag ggcgaatatc agtcctcgat gccggttacgctcgtttgat 26760 tcttcttcac aacgatgggc tcgaacttga ttaggcggtc tcgggaacggaaatcgcctg 26820 tacgccgtac tcacctggtg atcgtcttct tgctcttacc attgcggctatttgccgtct 26880 gctggttctg cacatcatac ctctcataac ccagatgcgt gtcctcccccaacctttcta 26940 gtcctttcat tgttagtgga tttgattgag tttacacgaa gtatttacgactcttggaac 27000 ccgggtgttt tttgcaagtg aaaaatgctg agttttgctg caaaaagtgccaggctccct 27060 tgccagccaa aacattactg cggaatgcgt tccattgcct gcttgaggcggaaactctcg 27120 cctgtgaacg agagaatatg cgcgtggtgc acgagacggt cgaccaaggctgaggtgagc 27180 ttagtatccc caaatatgga ggtccattgg ccaaactcca ggtactgggctgtgggtttt 27240 gcgcttgcga agggcggcgt tccagtcgct ctggtcggca tagcgtttggccgttcgcca 27300 gtgaatgccc acctaccttg cgatttcgct cactgagcag ccttctgtttcgcgtaaaaa 27360 agttgttgag gcattttcag catccttccc gtctccttcg tcaagttctcggcaaaccta 27420 acgataggag gattttaagg ggctgacaag tgcctttttt gcatctgacttcagcatttt 27480 tacgctgcaa ttctaggcat ttttagtatg caataaacac ccgggtctccatctctgaca 27540 tgagcctgcc gggcactcat ataatataaa aagcatcaga gctgaatgagtcggctgctg 27600 atgcttattt attggaccga acgatatctc tgcaagtaca agtacgaatggagcccggta 27660 ttgttgatac ccccgttttt ataaggatag actaagcagt tctttaatagactttaaatc 27720 tttcaggatg agtttatctg gtatccaaat gacaggaatt tgagttttattgattttgta 27780 attggttata atgaaatcga atttgaccgt attgcttggt tcggtttgaaggtttagaac 27840 gttacccaat tcattattta atttgtgata gatgaatttt ttccaattggttggtccgga 27900 taaaagcaat aaaacctttt ttttataatt tgacaggaga atagataaaatatgaaacgt 27960 gatgtggtag ctctcatctt cagataatct attttcattt atttctttattccatgcgac 28020 aataagaggg tgaatgatgt tgtagccctc tattaattca ttaggaatcgtcttaaatct 28080 accccaatag tattctaacg gtaaattgag atttttaaat atatagatcttgtggaaaga 28140 aaaatggatg ttatgtctta tttcttctat tactttggca ttcatatttacttcacttga 28200 aatcatgtca attaaagcaa ggtgcttatc gtaaagttgt ttatatgatccacgatagta 28260 attcttgatt ttactttttt cattatcatc cccctcggag atgaggaaaaaaaatagtgt 28320 aaaaaactta agttcatttt gctggaagct gaccttatag aaattttcaatggcagagac 28380 aatggcatag gtgcaatcta acttttgaga gctaaatatg tagtccatattaaaactttt 28440 ggaaataaaa tgcttgtata tatttctatt tacagacaca ttgatgagtatggttaatag 28500 attataatcg atctccattt tatagaaacg aatatagttc tcaattttttgttgcaagtc 28560 taggtccggt ttaaagattt ctttatactt ttgcacgtga aaaaaaagaaccatatagaa 28620 gtatctaata ttcagttcct ttccaattat ttggatcgtt gttctgaaattaaaatcaag 28680 gttaaaatgt ctaagtattt ttctgaaatt atttaatttt ttcttcaatgttaatttatc 28740 taagtacaat atttgagacc atttttccaa gctataatac ttctgattgaatattcccat 28800 taaaatgatg taaagttcac tgtttattat ataagtagct ataatagtggaaaggtgatc 28860 taaaggatct ttttttagaa cgtaaccttt ggaattaatg cttattatatcccaattatc 28920 tggaagatcc tgttttaatt gagaaatgtc acttataatc gttctgcttgtacattgcag 28980 cttacgggct aaagaactgg aagacacgat accttcgctg tcaattaaggattctaatat 29040 ttggattttt cgaataattg aatgattttg aattaagtga gcggtgaattcatccattta 29100 gtctccccct cgaaaagcga taactggtta aagtgcagat gtactatcctttgttcgaag 29160 ttgctagtaa tcaattctgc ggatctcttc aaagtgctcc aggtgctatggatttacgaa 29220 tttcccctaa acatatcttt ctaatgtttg tcttcttaat gaaaatctttattatttttc 29280 tccttttcac aatttaaatt tcaattagaa agtaacgtaa ttttggaatgaaaaacgata 29340 cataatttca ttaatagatg aaaatatgta tggtttttaa aatgtttgtaaaggtcagct 29400 taatgcggtt aatagaaggt taagagattt tattgtgcaa ggggggacgaagaaggatat 29460 aagatagaac atatccgaaa gggaggaggc taacgaaatt tgaaattatttgttgggtag 29520 tgtttgcaaa actcgaatta tatggtaatc tatagatggt caattatttgcaatgccaaa 29580 ggaaggtgag gctgtgagta ccgttgctgc gaagccacag aggttgggggagctaataca 29640 gtactatcgg cagaagaagg aattgagtct gtcgaagctg caagaagcggtcggcattga 29700 taaaggcagc ctgtcgagaa ttgaaaacag cgaggtcaaa cgccctgattttcgatccat 29760 cttgtcgatc gccgcggtat tggacattcc ccatgacgcc atcgtagaacagtacatcga 29820 gatcggacat aaatcggaag tcatatacac tattttacag aacgaattgacaaccctcga 29880 gcatccatcg ctcataccga aaattgccgc aaagtttctt gaagcgcccaatgaagacag 29940 tctggatgca gtagagaagt tgtaccgaac gataggctcc gtgaatcatccttccactca 30000 gttatcttta tacaccctca tcgtagacta ctcacgcgcc catggaatcatgccttacat 30060 tgccaaagga ttatttcgga agtacatgat tgaacgaaat gatttcagcaggttgaagga 30120 aacgtatcag gttggaaaaa atgtgctgga ctatgctaat tttttgagcgagaaagagcg 30180 gatacttcta tactatggct tgagtgttca cgcttatagt cttatgttttatcatgatgc 30240 cataaaattc ggtaattatg ttgtggaaaa tggggaagaa gaaccgctagcaaatgcaac 30300 tcataatgtt tgcaatgcct attaccattt ggggaattat gacgattgcaatacctatct 30360 cgaaaagtat agtcatttcc catatccttt cgtcaaagag aatgttaaattaatgactgc 30420 ctttcttaac gggaagaaag ggaatattga gtccgccatt actcagtttaataactgttt 30480 agataccctg tcctcatata atttgattca tgccgtaact gaattaatggaactatatct 30540 ccataaaaac gatcttgttg cagcagatca actccttatg tatgaagaacaaataattga 30600 gagcatcacc caaccacgaa ctacgccata taaaaggtca agattagcgcactactttcg 30660 catcaaagga caattgttaa ctcgtaaaca acatgaaaaa gacgctgttgatagtttctt 30720 gaaaagtaca ttagagtacg taaaaattgg tcgttttacg gaagcttttgaatcattgtc 30780 gtttgtgacg cattcaatga tacataatca gtcaatcatt aatagtgaaataattaaaaa 30840 ggttgataat attttacaaa taattgctgc aaaataatta aggaggaatatggtgccatg 30900 agaaaacgta aacttctttt cattgcctct cttctagttt ttggagcaatcagcatggag 30960 catattgttt catacatcga tgccccgtgg ataactaact tctaatgaatataacatctc 31020 ataacgctag tcatggcccc gccattcgtt ggtggggtaa catccttcgaaagcccgatt 31080 cttttactgg caatgttgct gcaattgtaa ggggcctcac tcactccaagtgctgtctct 31140 gcagatcctc tttggtttat gccatcggtc tatagcagca gtaactcgatcgacagataa 31200 aaatttgaaa attctcttcg aaaggagatt gacgcattaa gacgcctttatgggcgtttt 31260 tttgtttttt atgtataaag ttgttgcata ataatccaga atcaagtcatatttaccaac 31320 ttcatctata atcaggtcaa ataatacagt ttatggatga ggtgtggaaatgagccatca 31380 accggtatac aaaggtgact taatcccttt tacttatagc tatgagaaagctggctgttt 31440 aattgaaatc attgagagca ctcgacagga gcttatagta gcggccacggaaaaaaagtg 31500 tcttaccgac gaaactgttg taagattaag ccaaaagtta gatacatacctattggaatt 31560 tcaaagaagg agctgcggct agaagaatag gatgtgataa tccggattattaggggaagg 31620 tgaggctgtg agtatcggtt ctgtgaagcc taaaagcttg ggggagttaatcaagtatta 31680 tcgaaaaatg aatagcgaaa tcgcagatca gacattgaga ttgagcaaaaacctaaagtc 31740 attttctcaa tcttgcagaa cgaattggaa acactcgagc atccagcattaatacccgaa 31800 atagccgcca aatttcttga cttatcaaat ggtatggagg gggtaaaagagctttacaga 31860 gtaattgact cggtagaaga tacctccatt caattagctg tgtacaaccttattattgat 31920 tactctcgcg ctcatggaat gatgccctat attgctaagg gattataccgaaaatacatg 31980 atcgaacgaa atgatttcag caaattgaaa gaaacctatc aactaggtaagtgtgtattg 32040 gactatatac agtttctggg cgacgaagaa cgaattgtat tttattattcaataggagtt 32100 cacgctgaca gcttaatgaa ctatgatgac tctgtaccct atatgaaatatgtggtggag 32160 aacgataact cagaaaatgg tgcctataga gcgaatgctt atcttagtctatgcaattct 32220 tcctataata ctggtgatta caaagcaagc caggaatatt tagatgagtacagtaagtac 32280 tctttttcat atgtagccga taatgtgcat tttatgtcgg cttgtatagagggcaagatg 32340 gggaacgtgg atagttcgat ttcaaagtta cgttcttatt tacaaagttcatgggaagta 32400 tcccgttgaa aagggaacct aaatatattc aaacgccttg tggtgtctcagattgttgaa 32460 aaaccctgtc cttttctaaa ggtacagggt ttctcacatg aaaaggactcacgaaacgaa 32520 ccttccgatg aacccggcca cggttttttc gtacgtttcc cgatccatccggtatgcttc 32580 tccatgtccg gctttcggaa cgatgtatag ctctttctcg gcggggcaattttcgtaaac 32640 tttatgcacc atctccgtcg gcacaaaagt atcgttggcc ccatgaatgaagagagtcgg 32700 ggttttcgac tttttcacct gctccagtgc ggaggcttct ccgaaaaagtaccctgcccg 32760 tagcctggtc agcaggctgg tggtatccac gatcgggaat gccggaagatgatacatgcg 32820 ccgcagctgg aaggaaagct gatccttcac agaggtataa ccacagtcttcgacgatggc 32880 tttcacgttc ggcggcaaat tctcgccgct ggtcatcatc acggttgcccctcccatcga 32940 cacgccgtgg agaacgattt gtgaattcgt cccattcgtg tccaaaacccgctgaatcca 33000 tttcaaataa tccttacgct cgggccagcc gaaaccgata taatggccttcgctttcccc 33060 atgtccccga gcatcgggga gcaggatgtt gtagccccat ttctcgtggtacattctggc 33120 gtaaccgctc atttgcgttg cgttcccgga atatccgtgc gcgatgatgaccgttttgtc 33180 cgacggcttg gatgccggca aataatacgc cttcagatga atgccgtcatccgagtccat 33240 ctcccagcgc tcaaagcttt ggttgttcca ccattccttg tccgccagcgtggtttgctt 33300 tgattcctcc acctcaggac tggttttcag gtcgggattg tcgctcaaaaagtctttcga 33360 agcgcgcgca atcgccactt gataaaagta aaagctcccg gcggtaaggataataatgac 33420 aaatacaatc aaggaaataa agcctgtcac catttttttc ttcaattctgttctctccta 33480 gtaaactctc atgttagtta ttttaatata tcataatgat c 33521 23264 DNA Paenibacillus strain IDAS 1529 2 gatcacacgg ccggcgtattccggctcgga accgaagaat taacagaagc gcttcagcag 60 tccggttatc ggacagtctttgatattgca tctgaaaatc ttgcggaatt tcagaaaagc 120 aatccggaga ttccctcttccgacgcgaag gagattcatc aattagccgt ccagaggaca 180 gaaaacttat gcatgctttataaggcctgg caactgcaca atgatccggt cgtccagagc 240 cttcccaaat tatccgcggataccggcctg cgaggcatgc gtgccgcgtt ggagcggagt 300 cttggagggg gagccgattttggagacttg ttcccggagc gatcgccaga gggctatgcg 360 gaagcctcct ctatacagtcgcttttttcg ccgggacgtt accttacggt gctgtataaa 420 attgcgcagg atctccacgacccaaaagac aaactgcata ttgacaaccg ccgtccagat 480 ttgaagtcgc tgatcctcaataatgacaat atgaaccgtg aggtgtcttc cctggatatc 540 ctgctggatg tgctgcagtccgaaggctcc ggcacactga catccctgaa ggatacctac 600 tatccgatga cccttccctatgatgacgac cttgcgcaaa tcaatgccgt ggcggaggcg 660 cgctcatcca atttgctggggatctgggat accctgctgg acacgcagcg gacttccatc 720 ctgcaggatt ccgccgctgtccaccggata agcaagccgc ggcactcggc atacgtcaat 780 cagagagtct ccgatgatgaaccggtattg atcgcgggag aggaattcta cttggagacc 840 ggcggtgttg ccgacacgaccccgtctccg ccaacgaggg aagcgctttc cttgacgcca 900 aacagcttcc gtctgctggtcaaccccgag ccgacagcag acgacatcgc caatcactac 960 aacgttaaga ctcaagatcctgccgctctg gccgccgtct taaatgtggt cgatgacttt 1020 tgcctgaaaa ccggtttgagctttaatgag ttgctggact taacgatgca gaaggatgat 1080 gaatcgatcg gcagcgagtacaaaagccgg tttgtaaaat ttggcggcga ggccaatgtt 1140 ccggtttcaa cctatggagctgtatttctg acaggaacgg aagaaactcc gttgtgggta 1200 ggaaaaggag ctgtgataagccctgcagcg gacgcctatg ttcgtaatgg gacatatgca 1260 aacacgaatt atggatcagacactagtctt gttgtgaagc aggatgggtc tagtggatac 1320 agtagggaag catatatcaggtttgatttg acaggtcttt ccggagttgt ggaggaagct 1380 aaaatttctc taacaactagagcgaaacaa ttgtctagct taagacacca agctcatttg 1440 gtcagtgaca acagttgggatgaattgaaa atcacatgga ataacaaacc tgcaggagga 1500 gcgatcatcg caagctgggatgttcccgaa gttggtgaga atgtaaaggt tgatgtgacc 1560 cggcaagtaa atgatgcgctcgcaaacggt caagataaac tatcaattgt tattcgttct 1620 agtgcaaatt atggcagtctgggcgatgtc tcttatgcct ctagagaaca ccctgaaaaa 1680 gcctcacgac cttctatggaaatcaaggcg ataacgggtg ctggtttaaa ttttacggcg 1740 gataatgttg tagctctggcaggaagggcg gaaaagcttg tccggctggc gcgcagcacg 1800 ggactttcct ttgagcagttggattggctg attaccaata ccagccgtgc cgtaatcgaa 1860 catggtggag aactgattctggataagccg gtactggagt ctgtggccga attcacaagg 1920 ctccataagc gttatggcatcacagcggat atgttcgccg cgtttatcgg cgaagtcaat 1980 acgtatgctg aagcaggtaaagagagcttt tatcagacga ttttcagcac ggccgaccat 2040 tcggctgcct tacctttaggcgcaactttg caatttgagg tgagcaaaca ggatcgatat 2100 gaagcgattt gctgcggggccatgggggtg accgccgatg agttctctcg tatcggcaaa 2160 tactgctttg gcgacaacgcgcagcaagtt accgccaatg aaacaaccgt tgcgcagctt 2220 tatcgtttag gccgaattcctcacatgctt ggattgcgtt ttaccgaggc ggagctgttg 2280 tggaaattga tggctggcggcgaggatacc ttgctccgca cgattggcgc gaagcctcgc 2340 agtttacaag ccttagagattattcgccgt actgaggtcc ttttggactg gatggatgct 2400 catcagcttg atgttgtctccctgcaagcc atggttacca atcggtacag cggcacagcc 2460 acgccggagc tgtacaactttttggcacag gtgcaccaat ccacaagcag tgccgcgaac 2520 gtgtccaaag cggatgctcaggataccctg cccgcggaca agctgttccg ggccttggcg 2580 gtaggcttca acctgaaggccaacgtgatg gcgcaggtca ttgactggtt ggacaaaacc 2640 gacggagcgt ttacgctgcgggctttctgg gacaagcttc aagcgtattt cagcgccgat 2700 catgaagaag aactgacggccttggaagga gaagccgact tgctgcagtg gtgccaacag 2760 atcagccagt atgcgctcattgtccgctgg tgcgggttaa gcgatcagga tctggcgctg 2820 ctgaccgggc atcccgggcagcttctgtcc ggacaacata cggtgccggt accctcgctg 2880 catctcctgc tggtgctgacccgcctgaag gaatggcagc agcgcgtcca ggtttccagc 2940 gaggaggcca tgcgctattttgcccaggcc gatgcgccaa ccgtcacacg cgatgctgcg 3000 gtcaagctgc ttgcccgtatccatggctgg aatgaacagg ataccgcctc gatgaatgac 3060 tacctgctgg gagagaacgaatatcctaag aactttgagc agatctttac tttggaaagc 3120 tgggtcaacc tgggccgtcaactgaatgtt ggcagccgaa cgttgggaga gctggttgac 3180 atgtcagaag aggatgataccgcggaaaac acggatttga ttatctcggt cgcccaaagc 3240 ctgatggctg cggtgcaggcctga 3264 3 1087 PRT Paenibacillus strain IDAS 1529 3 Asp His Thr AlaGly Val Phe Arg Leu Gly Thr Glu Glu Leu Thr Glu 1 5 10 15 Ala Leu GlnGln Ser Gly Tyr Arg Thr Val Phe Asp Ile Ala Ser Glu 20 25 30 Asn Leu AlaGlu Phe Gln Lys Ser Asn Pro Glu Ile Pro Ser Ser Asp 35 40 45 Ala Lys GluIle His Gln Leu Ala Val Gln Arg Thr Glu Asn Leu Cys 50 55 60 Met Leu TyrLys Ala Trp Gln Leu His Asn Asp Pro Val Val Gln Ser 65 70 75 80 Leu ProLys Leu Ser Ala Asp Thr Gly Leu Arg Gly Met Arg Ala Ala 85 90 95 Leu GluArg Ser Leu Gly Gly Gly Ala Asp Phe Gly Asp Leu Phe Pro 100 105 110 GluArg Ser Pro Glu Gly Tyr Ala Glu Ala Ser Ser Ile Gln Ser Leu 115 120 125Phe Ser Pro Gly Arg Tyr Leu Thr Val Leu Tyr Lys Ile Ala Gln Asp 130 135140 Leu His Asp Pro Lys Asp Lys Leu His Ile Asp Asn Arg Arg Pro Asp 145150 155 160 Leu Lys Ser Leu Ile Leu Asn Asn Asp Asn Met Asn Arg Glu ValSer 165 170 175 Ser Leu Asp Ile Leu Leu Asp Val Leu Gln Ser Glu Gly SerGly Thr 180 185 190 Leu Thr Ser Leu Lys Asp Thr Tyr Tyr Pro Met Thr LeuPro Tyr Asp 195 200 205 Asp Asp Leu Ala Gln Ile Asn Ala Val Ala Glu AlaArg Ser Ser Asn 210 215 220 Leu Leu Gly Ile Trp Asp Thr Leu Leu Asp ThrGln Arg Thr Ser Ile 225 230 235 240 Leu Gln Asp Ser Ala Ala Val His ArgIle Ser Lys Pro Arg His Ser 245 250 255 Ala Tyr Val Asn Gln Arg Val SerAsp Asp Glu Pro Val Leu Ile Ala 260 265 270 Gly Glu Glu Phe Tyr Leu GluThr Gly Gly Val Ala Asp Thr Thr Pro 275 280 285 Ser Pro Pro Thr Arg GluAla Leu Ser Leu Thr Pro Asn Ser Phe Arg 290 295 300 Leu Leu Val Asn ProGlu Pro Thr Ala Asp Asp Ile Ala Asn His Tyr 305 310 315 320 Asn Val LysThr Gln Asp Pro Ala Ala Leu Ala Ala Val Leu Asn Val 325 330 335 Val AspAsp Phe Cys Leu Lys Thr Gly Leu Ser Phe Asn Glu Leu Leu 340 345 350 AspLeu Thr Met Gln Lys Asp Asp Glu Ser Ile Gly Ser Glu Tyr Lys 355 360 365Ser Arg Phe Val Lys Phe Gly Gly Glu Ala Asn Val Pro Val Ser Thr 370 375380 Tyr Gly Ala Val Phe Leu Thr Gly Thr Glu Glu Thr Pro Leu Trp Val 385390 395 400 Gly Lys Gly Ala Val Ile Ser Pro Ala Ala Asp Ala Tyr Val ArgAsn 405 410 415 Gly Thr Tyr Ala Asn Thr Asn Tyr Gly Ser Asp Thr Ser LeuVal Val 420 425 430 Lys Gln Asp Gly Ser Ser Gly Tyr Ser Arg Glu Ala TyrIle Arg Phe 435 440 445 Asp Leu Thr Gly Leu Ser Gly Val Val Glu Glu AlaLys Ile Ser Leu 450 455 460 Thr Thr Arg Ala Lys Gln Leu Ser Ser Leu ArgHis Gln Ala His Leu 465 470 475 480 Val Ser Asp Asn Ser Trp Asp Glu LeuLys Ile Thr Trp Asn Asn Lys 485 490 495 Pro Ala Gly Gly Ala Ile Ile AlaSer Trp Asp Val Pro Glu Val Gly 500 505 510 Glu Asn Val Lys Val Asp ValThr Arg Gln Val Asn Asp Ala Leu Ala 515 520 525 Asn Gly Gln Asp Lys LeuSer Ile Val Ile Arg Ser Ser Ala Asn Tyr 530 535 540 Gly Ser Leu Gly AspVal Ser Tyr Ala Ser Arg Glu His Pro Glu Lys 545 550 555 560 Ala Ser ArgPro Ser Met Glu Ile Lys Ala Ile Thr Gly Ala Gly Leu 565 570 575 Asn PheThr Ala Asp Asn Val Val Ala Leu Ala Gly Arg Ala Glu Lys 580 585 590 LeuVal Arg Leu Ala Arg Ser Thr Gly Leu Ser Phe Glu Gln Leu Asp 595 600 605Trp Leu Ile Thr Asn Thr Ser Arg Ala Val Ile Glu His Gly Gly Glu 610 615620 Leu Ile Leu Asp Lys Pro Val Leu Glu Ser Val Ala Glu Phe Thr Arg 625630 635 640 Leu His Lys Arg Tyr Gly Ile Thr Ala Asp Met Phe Ala Ala PheIle 645 650 655 Gly Glu Val Asn Thr Tyr Ala Glu Ala Gly Lys Glu Ser PheTyr Gln 660 665 670 Thr Ile Phe Ser Thr Ala Asp His Ser Ala Ala Leu ProLeu Gly Ala 675 680 685 Thr Leu Gln Phe Glu Val Ser Lys Gln Asp Arg TyrGlu Ala Ile Cys 690 695 700 Cys Gly Ala Met Gly Val Thr Ala Asp Glu PheSer Arg Ile Gly Lys 705 710 715 720 Tyr Cys Phe Gly Asp Asn Ala Gln GlnVal Thr Ala Asn Glu Thr Thr 725 730 735 Val Ala Gln Leu Tyr Arg Leu GlyArg Ile Pro His Met Leu Gly Leu 740 745 750 Arg Phe Thr Glu Ala Glu LeuLeu Trp Lys Leu Met Ala Gly Gly Glu 755 760 765 Asp Thr Leu Leu Arg ThrIle Gly Ala Lys Pro Arg Ser Leu Gln Ala 770 775 780 Leu Glu Ile Ile ArgArg Thr Glu Val Leu Leu Asp Trp Met Asp Ala 785 790 795 800 His Gln LeuAsp Val Val Ser Leu Gln Ala Met Val Thr Asn Arg Tyr 805 810 815 Ser GlyThr Ala Thr Pro Glu Leu Tyr Asn Phe Leu Ala Gln Val His 820 825 830 GlnSer Thr Ser Ser Ala Ala Asn Val Ser Lys Ala Asp Ala Gln Asp 835 840 845Thr Leu Pro Ala Asp Lys Leu Phe Arg Ala Leu Ala Val Gly Phe Asn 850 855860 Leu Lys Ala Asn Val Met Ala Gln Val Ile Asp Trp Leu Asp Lys Thr 865870 875 880 Asp Gly Ala Phe Thr Leu Arg Ala Phe Trp Asp Lys Leu Gln AlaTyr 885 890 895 Phe Ser Ala Asp His Glu Glu Glu Leu Thr Ala Leu Glu GlyGlu Ala 900 905 910 Asp Leu Leu Gln Trp Cys Gln Gln Ile Ser Gln Tyr AlaLeu Ile Val 915 920 925 Arg Trp Cys Gly Leu Ser Asp Gln Asp Leu Ala LeuLeu Thr Gly His 930 935 940 Pro Gly Gln Leu Leu Ser Gly Gln His Thr ValPro Val Pro Ser Leu 945 950 955 960 His Leu Leu Leu Val Leu Thr Arg LeuLys Glu Trp Gln Gln Arg Val 965 970 975 Gln Val Ser Ser Glu Glu Ala MetArg Tyr Phe Ala Gln Ala Asp Ala 980 985 990 Pro Thr Val Thr Arg Asp AlaAla Val Lys Leu Leu Ala Arg Ile His 995 1000 1005 Gly Trp Asn Glu GlnAsp Thr Ala Ser Met Asn Asp Tyr Leu Leu 1010 1015 1020 Gly Glu Asn GluTyr Pro Lys Asn Phe Glu Gln Ile Phe Thr Leu 1025 1030 1035 Glu Ser TrpVal Asn Leu Gly Arg Gln Leu Asn Val Gly Ser Arg 1040 1045 1050 Thr LeuGly Glu Leu Val Asp Met Ser Glu Glu Asp Asp Thr Ala 1055 1060 1065 GluAsn Thr Asp Leu Ile Ile Ser Val Ala Gln Ser Leu Met Ala 1070 1075 1080Ala Val Gln Ala 1085 4 3618 DNA Paenibacillus strain IDAS 1529 4atgaccaagg aaggtggtaa gaatatgtct acttcaaccc tgttgcaatt gattaaggaa 60tcccgccggg atgcgttggt caaccattat atcgccaaca atgtcccgag agagcttacg 120gataagatta cagacgcaga cagcctgtat gagtatttgc tgctggatac caagatcagt 180gaactcgtaa aaacatcgcc gatagctgag gccattagca gcgttcagtt atacatgaac 240cgatgcgtgg aaggctatga aggcaagctg actccggaag gcaacagcca tttcgggccg 300ggaaaattcc tgaataattg ggatacctat aacaagcgtt attccacttg ggccggcaag 360gaacgtctga aatattatgc aggcagttat attgacccgt ccttgcgcta taacaaaacg 420gatccgttcc tgaacctgga acagaatatc agccagggaa gaatcaccga tgacaccgta 480aagaacgcgc tgcaacacta cctgactgaa tatgaagtgt tggcggattt ggaatatatc 540agcgtaaata aaggcgccga tgaaagtgta ttattcttcg taggccgcac caaaacaatg 600ccatacgaat attactggcg ccgattaacg ttgaaaaagg acaataacaa taaactggtg 660cctgccatct ggtctcaatg gaaaaaaata actgccaata tcggcgaagc agttaataat 720tatgtggtgc ttcactggca taataaccgc ttacatgtac aatggggttc tacagagaaa 780acacaaaatg atgacggaga acccattgag aaacgatatt tgaatgactg gttcatggat 840aagtccagtg tctggtcttc attccgaaag gtttcatata tagaaaatag ttttacttat 900actgagggca tcattgattc aagaaatatt actatagctg gaaatcaact gttctgtgat 960gattcaaata cttttaaggc aacaataacg gcacttccat ttgaccaaat acgtgtttac 1020ttagaaaaga tttacggtac aggcggcagc atcacggtta ctggagaaaa taaaggctat 1080attattaagg tgggggagcc aagagaagtc agtttctctc ctaatacgtt actagatgta 1140ttcataggta gtaatgcaag ccctcgagac ccatatttca aagctacatt taatagagaa 1200gctctccaaa attcatacgg ctcaattaaa ataaatcaat acacccctcc ttctggaagc 1260aatatcaaag gtcctatcga ccttaccctg aaaaataaca tcgacctgtc ggcgttgttg 1320gaagagagcc ttgacgtact gttcgactat accattcagg ggaataacca attgggcggc 1380ttagaggcct ttaacgggcc ttacggactt tatttgtggg aaatcttcct ccatgttcca 1440tttttaatgg cggttcgctt ccacaccgag cagcggtatg agttggcgga acgatggttt 1500aaattcattt tcaacagcgc aggttaccgt gatggctacg gcaatctgct gacggatgac 1560aaaggcaacg tgcgctactg gaacgtcgtg cctctgcagg aggatacgga gtgggatgac 1620acgttgtccc tggcaacgac cgacccggac gagattgcga tggccgaccc gatgcaatac 1680aagctggcta tctttattca caccttggac ttcttgatca gccgcggcga cagcttgtac 1740cggatgctgg agcgggatac cttgaccgaa gcgaagatgt attacattca ggccagccaa 1800ctgcttgggc ctcgtcccga gatccggatc aatcacagct ggcctgatcc gaccctgcaa 1860agcgaagcgg acgcggtaac cgccgtgccg acgcgaagcg attcgccggc agcgccaatt 1920ctcgccttgc gagcgcttct gaatgcggaa aacgggcatt tcctgccgcc ttataatgat 1980gaactattag ctttctggga taaaatcgac ctgcgtctct acaatttacg ccacaatctg 2040agcctggacg gtcagccgct tcatttgccg ctctttaccg aaccggtcaa tcctcgtgag 2100ctgcaggttc agcatggggc aggcgatgga ttagggggaa gcgccggttc cgtccaaagc 2160cgtcaaagtg tctatcgttt tcctctggtc atcgataagg cgcgcaatgc cgcgagtagt 2220gttatccaat tcgggaatgc cctggaaaac gcgctgacaa agcaggacag cgaggccatg 2280actatgctgt tgcaatccca gcagcagatt gtcctgcagc aaacccgcga tattcaggag 2340aagaacctgg cctcgctgca agcaagtctg gaagcaacga tgacagccaa agcgggcgcg 2400aaatcccgaa agacccattt tgccggcctg gcggataact ggatgtcgca taatgaaacc 2460gcctcacttg cactgcgtac cactgcggga attatcaata caagctcgac cgtgccaatc 2520gctatcactg gcggcttgga tatggctccg aacatttttg gtttcgcagt tggaggttcc 2580cgctggggag cagccagcgc ggctgtagcc caaggattgc aaatcgccgc cggcgtaatg 2640gaacagacgg ccaatatcat cgatatcagc gaaagctacc gccggcgccg ggaggattgg 2700ctgctgcagc gggatgttgc cgagaatgaa gcggcgcagt tggattcgca gattgcggcc 2760ctgcgggaac agatggatat ggcgcgaaaa caacttgcgc tggcggagac ggaacaggca 2820cacgcgcaag cggtctacga gctgctaagc acccgtttta cgaatcaagc tttgtataac 2880tggatggccg gacgtctgtc gtctctatac tatcaaatgt atgacgccgc attgccgctc 2940tgcttgatgg ccaaacaggc tttagagaaa gaaatcggca atgataaaac ggttggaatc 3000ttcaccctcc cggcctggaa tgatttgtat cagggattgc tagcgggcga ggcgctgctg 3060ctcgagcttc agaagctgga gaatctgtgg ctggaggagg acaagcgcgg aatggaagct 3120gtaagaacgg tatctttaga tacccttctc cgcaaagaaa agccagaatc cggttttgca 3180gatttcgtca aggaagttct ggacggaaag acgcctgacc ctgtaagcgg agttagcgta 3240cagctgcaaa acaatatttt cagtgcaacc cttgacctgt ccacccttgg cctggatcgc 3300ttttacaacc aagcggaaaa ggcccacagg atcaaaaacc tgtcggttac cttacccgcg 3360ctattgggac cttatcagga tattgcggca accttatcgc taggtggcga gaccgttgcg 3420ctttcccatg gcgtggatga cagcggcttg tttatcacgg atctcaacga cagccgtttc 3480ctgcctttcg agggtatgga tcctttatcc ggcacactcg ttctgtcgat actccatgcc 3540gggcaagacg gtgaccagcg cctcctgctg gaaagcctga acgacgtcat cttccacatt 3600cgatatgtca tgaaatag 3618 5 1205 PRT Paenibacillus strain IDAS 1529 5 MetThr Lys Glu Gly Gly Lys Asn Met Ser Thr Ser Thr Leu Leu Gln 1 5 10 15Leu Ile Lys Glu Ser Arg Arg Asp Ala Leu Val Asn His Tyr Ile Ala 20 25 30Asn Asn Val Pro Arg Glu Leu Thr Asp Lys Ile Thr Asp Ala Asp Ser 35 40 45Leu Tyr Glu Tyr Leu Leu Leu Asp Thr Lys Ile Ser Glu Leu Val Lys 50 55 60Thr Ser Pro Ile Ala Glu Ala Ile Ser Ser Val Gln Leu Tyr Met Asn 65 70 7580 Arg Cys Val Glu Gly Tyr Glu Gly Lys Leu Thr Pro Glu Gly Asn Ser 85 9095 His Phe Gly Pro Gly Lys Phe Leu Asn Asn Trp Asp Thr Tyr Asn Lys 100105 110 Arg Tyr Ser Thr Trp Ala Gly Lys Glu Arg Leu Lys Tyr Tyr Ala Gly115 120 125 Ser Tyr Ile Asp Pro Ser Leu Arg Tyr Asn Lys Thr Asp Pro PheLeu 130 135 140 Asn Leu Glu Gln Asn Ile Ser Gln Gly Arg Ile Thr Asp AspThr Val 145 150 155 160 Lys Asn Ala Leu Gln His Tyr Leu Thr Glu Tyr GluVal Leu Ala Asp 165 170 175 Leu Glu Tyr Ile Ser Val Asn Lys Gly Ala AspGlu Ser Val Leu Phe 180 185 190 Phe Val Gly Arg Thr Lys Thr Met Pro TyrGlu Tyr Tyr Trp Arg Arg 195 200 205 Leu Thr Leu Lys Lys Asp Asn Asn AsnLys Leu Val Pro Ala Ile Trp 210 215 220 Ser Gln Trp Lys Lys Ile Thr AlaAsn Ile Gly Glu Ala Val Asn Asn 225 230 235 240 Tyr Val Val Leu His TrpHis Asn Asn Arg Leu His Val Gln Trp Gly 245 250 255 Ser Thr Glu Lys ThrGln Asn Asp Asp Gly Glu Pro Ile Glu Lys Arg 260 265 270 Tyr Leu Asn AspTrp Phe Met Asp Lys Ser Ser Val Trp Ser Ser Phe 275 280 285 Arg Lys ValSer Tyr Ile Glu Asn Ser Phe Thr Tyr Thr Glu Gly Ile 290 295 300 Ile AspSer Arg Asn Ile Thr Ile Ala Gly Asn Gln Leu Phe Cys Asp 305 310 315 320Asp Ser Asn Thr Phe Lys Ala Thr Ile Thr Ala Leu Pro Phe Asp Gln 325 330335 Ile Arg Val Tyr Leu Glu Lys Ile Tyr Gly Thr Gly Gly Ser Ile Thr 340345 350 Val Thr Gly Glu Asn Lys Gly Tyr Ile Ile Lys Val Gly Glu Pro Arg355 360 365 Glu Val Ser Phe Ser Pro Asn Thr Leu Leu Asp Val Phe Ile GlySer 370 375 380 Asn Ala Ser Pro Arg Asp Pro Tyr Phe Lys Ala Thr Phe AsnArg Glu 385 390 395 400 Ala Leu Gln Asn Ser Tyr Gly Ser Ile Lys Ile AsnGln Tyr Thr Pro 405 410 415 Pro Ser Gly Ser Asn Ile Lys Gly Pro Ile AspLeu Thr Leu Lys Asn 420 425 430 Asn Ile Asp Leu Ser Ala Leu Leu Glu GluSer Leu Asp Val Leu Phe 435 440 445 Asp Tyr Thr Ile Gln Gly Asn Asn GlnLeu Gly Gly Leu Glu Ala Phe 450 455 460 Asn Gly Pro Tyr Gly Leu Tyr LeuTrp Glu Ile Phe Leu His Val Pro 465 470 475 480 Phe Leu Met Ala Val ArgPhe His Thr Glu Gln Arg Tyr Glu Leu Ala 485 490 495 Glu Arg Trp Phe LysPhe Ile Phe Asn Ser Ala Gly Tyr Arg Asp Gly 500 505 510 Tyr Gly Asn LeuLeu Thr Asp Asp Lys Gly Asn Val Arg Tyr Trp Asn 515 520 525 Val Val ProLeu Gln Glu Asp Thr Glu Trp Asp Asp Thr Leu Ser Leu 530 535 540 Ala ThrThr Asp Pro Asp Glu Ile Ala Met Ala Asp Pro Met Gln Tyr 545 550 555 560Lys Leu Ala Ile Phe Ile His Thr Leu Asp Phe Leu Ile Ser Arg Gly 565 570575 Asp Ser Leu Tyr Arg Met Leu Glu Arg Asp Thr Leu Thr Glu Ala Lys 580585 590 Met Tyr Tyr Ile Gln Ala Ser Gln Leu Leu Gly Pro Arg Pro Glu Ile595 600 605 Arg Ile Asn His Ser Trp Pro Asp Pro Thr Leu Gln Ser Glu AlaAsp 610 615 620 Ala Val Thr Ala Val Pro Thr Arg Ser Asp Ser Pro Ala AlaPro Ile 625 630 635 640 Leu Ala Leu Arg Ala Leu Leu Asn Ala Glu Asn GlyHis Phe Leu Pro 645 650 655 Pro Tyr Asn Asp Glu Leu Leu Ala Phe Trp AspLys Ile Asp Leu Arg 660 665 670 Leu Tyr Asn Leu Arg His Asn Leu Ser LeuAsp Gly Gln Pro Leu His 675 680 685 Leu Pro Leu Phe Thr Glu Pro Val AsnPro Arg Glu Leu Gln Val Gln 690 695 700 His Gly Ala Gly Asp Gly Leu GlyGly Ser Ala Gly Ser Val Gln Ser 705 710 715 720 Arg Gln Ser Val Tyr ArgPhe Pro Leu Val Ile Asp Lys Ala Arg Asn 725 730 735 Ala Ala Ser Ser ValIle Gln Phe Gly Asn Ala Leu Glu Asn Ala Leu 740 745 750 Thr Lys Gln AspSer Glu Ala Met Thr Met Leu Leu Gln Ser Gln Gln 755 760 765 Gln Ile ValLeu Gln Gln Thr Arg Asp Ile Gln Glu Lys Asn Leu Ala 770 775 780 Ser LeuGln Ala Ser Leu Glu Ala Thr Met Thr Ala Lys Ala Gly Ala 785 790 795 800Lys Ser Arg Lys Thr His Phe Ala Gly Leu Ala Asp Asn Trp Met Ser 805 810815 His Asn Glu Thr Ala Ser Leu Ala Leu Arg Thr Thr Ala Gly Ile Ile 820825 830 Asn Thr Ser Ser Thr Val Pro Ile Ala Ile Thr Gly Gly Leu Asp Met835 840 845 Ala Pro Asn Ile Phe Gly Phe Ala Val Gly Gly Ser Arg Trp GlyAla 850 855 860 Ala Ser Ala Ala Val Ala Gln Gly Leu Gln Ile Ala Ala GlyVal Met 865 870 875 880 Glu Gln Thr Ala Asn Ile Ile Asp Ile Ser Glu SerTyr Arg Arg Arg 885 890 895 Arg Glu Asp Trp Leu Leu Gln Arg Asp Val AlaGlu Asn Glu Ala Ala 900 905 910 Gln Leu Asp Ser Gln Ile Ala Ala Leu ArgGlu Gln Met Asp Met Ala 915 920 925 Arg Lys Gln Leu Ala Leu Ala Glu ThrGlu Gln Ala His Ala Gln Ala 930 935 940 Val Tyr Glu Leu Leu Ser Thr ArgPhe Thr Asn Gln Ala Leu Tyr Asn 945 950 955 960 Trp Met Ala Gly Arg LeuSer Ser Leu Tyr Tyr Gln Met Tyr Asp Ala 965 970 975 Ala Leu Pro Leu CysLeu Met Ala Lys Gln Ala Leu Glu Lys Glu Ile 980 985 990 Gly Asn Asp LysThr Val Gly Ile Phe Thr Leu Pro Ala Trp Asn Asp 995 1000 1005 Leu TyrGln Gly Leu Leu Ala Gly Glu Ala Leu Leu Leu Glu Leu 1010 1015 1020 GlnLys Leu Glu Asn Leu Trp Leu Glu Glu Asp Lys Arg Gly Met 1025 1030 1035Glu Ala Val Arg Thr Val Ser Leu Asp Thr Leu Leu Arg Lys Glu 1040 10451050 Lys Pro Glu Ser Gly Phe Ala Asp Phe Val Lys Glu Val Leu Asp 10551060 1065 Gly Lys Thr Pro Asp Pro Val Ser Gly Val Ser Val Gln Leu Gln1070 1075 1080 Asn Asn Ile Phe Ser Ala Thr Leu Asp Leu Ser Thr Leu GlyLeu 1085 1090 1095 Asp Arg Phe Tyr Asn Gln Ala Glu Lys Ala His Arg IleLys Asn 1100 1105 1110 Leu Ser Val Thr Leu Pro Ala Leu Leu Gly Pro TyrGln Asp Ile 1115 1120 1125 Ala Ala Thr Leu Ser Leu Gly Gly Glu Thr ValAla Leu Ser His 1130 1135 1140 Gly Val Asp Asp Ser Gly Leu Phe Ile ThrAsp Leu Asn Asp Ser 1145 1150 1155 Arg Phe Leu Pro Phe Glu Gly Met AspPro Leu Ser Gly Thr Leu 1160 1165 1170 Val Leu Ser Ile Leu His Ala GlyGln Asp Gly Asp Gln Arg Leu 1175 1180 1185 Leu Leu Glu Ser Leu Asn AspVal Ile Phe His Ile Arg Tyr Val 1190 1195 1200 Met Lys 1205 6 3300 DNAPaenibacillus strain IDAS 1529 6 atggtgtcaa caacagacaa cacggccggcgtattccggc tcggaaccga agaattaaca 60 gaagcgctta agcagtccgg ttatcggaccgtctttgata ttgtatctga caatcttgcg 120 gaatttcaga aaaacaatcc ggagattccctcttctgacg cgaaggagat tcatcaatta 180 gccgtccaga ggacagaaaa cttatgcatgctttataagg cctggcagct gcacaatgat 240 ccggttgtcc agagccttcc caaattatccgcggataccg gcctgcaagg catgcgtgcc 300 gcgttggagc ggagtcttgg aggcggagccgattttggag acttgttccc ggagcgatcg 360 ccagagggct atgcggaagc ctcctctatacagtcgcttt tctcgccggg acgttacctg 420 acggtgctgt ataaaattgc gcgggatctccacgacccaa aagataaact gcatattgac 480 aaccgccgtc cagatttgaa gtcgctgatcctcaataatg acaatatgaa ccgagaggta 540 tcttctctgg atatccttct ggatgtgctgcagcccgaag gctctgacac gctgacatcc 600 ttgaaggata cctaccatcc gatgacccttccctatgatg acgaccttgc gcaaatcaat 660 gccgtggcgg aggcgcgttc atctaatttgctggggattt gggataccct gctggacacg 720 cagcggactt ccatcctgca gaattccgccgctgcccgcc ggataagcaa ggcgcggcac 780 tcggcatacg ccaatcagaa agcctccaatgatgagccgg tattcatcac gggagaggaa 840 atctacctgg aaaccggagg taaacggctttttctggcgc ataaactcga gataggttca 900 actattagcg ctaaaatcaa cattggaccgccgcaagcgg ccgatatcgc gccggcaaag 960 ttgcaactcg tatattacgg cagaggcggcagagggaact acttcctgcg cgtggcagac 1020 gatgtgtccc tcggtggaaa gctgctgaccaattgttatc tgaccagcga tgacggacag 1080 agcaacaata ttagcgggcc atactgcctaatgatcaacc gaggcaccgg cagcatgcct 1140 agcgggactc accttccagt tcagattgaaagagtgaccg atacatccat ccgcattttt 1200 gtgccggatc acggctattt ggggctaggcgaaagccttg ccagcaactg gaatgaaccg 1260 ttggcgctga atctgggctt ggatgaagcgttgaccttta ccttgagaaa gaaggagacg 1320 ggaaatgaca ccatttccat aatcgacatgctgccgccgg tagcgaacac gactccgtct 1380 ccgccgacga gggaaacgct ttccttgacgccaaacagct tccgtctgct ggtcaaccct 1440 gagccgacag cggaggacat cgccaagcactacaacgtca cgacggtaac ccgggctcct 1500 gccgatctgg cctccgcctt aaatgttgtcgatgatttct gcttgaaaac cggtttgagc 1560 tttaacgaat tgctggattt aaccatgcagaaggattatc agtcaaaaag cagtgagtac 1620 aaaagccgat ttgtaaaatt cggcggcggggagaatgttc cggtatcaag ctatggcgca 1680 gcctttctga caggagcgga agatactcctttgtgggtga aacagtataa cagcgtgggg 1740 actgcaacaa gcacccctgt tttaaactttacgccagata atgttgtggc tttggcagga 1800 agggcggaaa agcttgtccg gctgatgcgcagcacgggtc tttcctttga gcagttggat 1860 tggctgattg ccaatgccag ccgtgccgttatcgaacacg gtggagagct ttttctggat 1920 aagccggtac tggaagctgt ggccgaattcacaaggctca ataagcgtta tggcgtcaca 1980 tcggatatgt tcgccgcgtt tatcggcgaagtcaatacgt atacagaagc gggcaaggac 2040 agcttttatc aggcgagttt cagcacggccgaccattcgg ctaccttacc tttgggcgct 2100 tctttgcaac ttgaggtgag caagcaggatcgatatgaag cgatttgctg cggggctatg 2160 ggggtgaccg ccgatgagtt ctcccgtatcggcaaatact gctttgggga taaagcacag 2220 caaatcacgg ccaatgaaac aaccgttgcccagctttatc gtttaggccg aattcctcat 2280 atgctaggct tgcgttttac cgaggcagagctgttgtgga aattgatggc tgggggcgag 2340 gataccttgc tccgcacgat tggcgcgaaccctcgcagtt tagaagcgtt agagattatt 2400 cgccggacgg aggtcctttt ggactggatggatgcccatc agctggatgt tgtctccctg 2460 caagccatgg ttaccaatcg gtacagcggcacagccacgc cggagctgta caattttttg 2520 gcacaggtgc atcaatccgc aagcagtgccgcgaacgtgg ccagagcgga tggtcaggat 2580 acgttgcctg cggacaagct gctccgggcattggcggcgg gcttcaaact gaaagccaac 2640 gtgatggcgc gagtaatcga ctggatggacaaaaccaata aagcgtttac gctgcgggct 2700 ttctgggaca agcttcaagc gtatttcagcgccgatcatg aagaagaact gaccgccctg 2760 gaaggagaag ccgcaatgct gcagtggtgccagcagatca gccagtatgc gctcattgtc 2820 cgctggtgcg ggttaagcga gcaggatctggcgctgctga ccgggaatcc ggagcagctt 2880 ctggacggac aacatacggt gcccgtaccctcgctgcatc tcctgctggt gctgacccgc 2940 ctgaaggaat ggcagcagcg cgtccaggtttccagcgagg aggctatgcg ctattttgcc 3000 caggccgatt cgccaaccgt cacgcgcgacgatgcggtta atctgcttgc ccgtatccat 3060 ggctggaatg aagcggatac cgtctcgatgaatgactacc tgctgggaga gaacgaatat 3120 cctaagaact ttgatcagat ctttgcactggaaagctggg tcaacctggg ccgtcaactg 3180 aacgtgggca gcagaacgct gggagagctggttgacatgg ctgaagagga taaaaccgcg 3240 gaaaacatgg atctgattac ttcggtggcccatagcctga tggctgcagc gaaagcctga 3300 7 1099 PRT Paenibacillus strainIDAS 1529 7 Met Val Ser Thr Thr Asp Asn Thr Ala Gly Val Phe Arg Leu GlyThr 1 5 10 15 Glu Glu Leu Thr Glu Ala Leu Lys Gln Ser Gly Tyr Arg ThrVal Phe 20 25 30 Asp Ile Val Ser Asp Asn Leu Ala Glu Phe Gln Lys Asn AsnPro Glu 35 40 45 Ile Pro Ser Ser Asp Ala Lys Glu Ile His Gln Leu Ala ValGln Arg 50 55 60 Thr Glu Asn Leu Cys Met Leu Tyr Lys Ala Trp Gln Leu HisAsn Asp 65 70 75 80 Pro Val Val Gln Ser Leu Pro Lys Leu Ser Ala Asp ThrGly Leu Gln 85 90 95 Gly Met Arg Ala Ala Leu Glu Arg Ser Leu Gly Gly GlyAla Asp Phe 100 105 110 Gly Asp Leu Phe Pro Glu Arg Ser Pro Glu Gly TyrAla Glu Ala Ser 115 120 125 Ser Ile Gln Ser Leu Phe Ser Pro Gly Arg TyrLeu Thr Val Leu Tyr 130 135 140 Lys Ile Ala Arg Asp Leu His Asp Pro LysAsp Lys Leu His Ile Asp 145 150 155 160 Asn Arg Arg Pro Asp Leu Lys SerLeu Ile Leu Asn Asn Asp Asn Met 165 170 175 Asn Arg Glu Val Ser Ser LeuAsp Ile Leu Leu Asp Val Leu Gln Pro 180 185 190 Glu Gly Ser Asp Thr LeuThr Ser Leu Lys Asp Thr Tyr His Pro Met 195 200 205 Thr Leu Pro Tyr AspAsp Asp Leu Ala Gln Ile Asn Ala Val Ala Glu 210 215 220 Ala Arg Ser SerAsn Leu Leu Gly Ile Trp Asp Thr Leu Leu Asp Thr 225 230 235 240 Gln ArgThr Ser Ile Leu Gln Asn Ser Ala Ala Ala Arg Arg Ile Ser 245 250 255 LysAla Arg His Ser Ala Tyr Ala Asn Gln Lys Ala Ser Asn Asp Glu 260 265 270Pro Val Phe Ile Thr Gly Glu Glu Ile Tyr Leu Glu Thr Gly Gly Lys 275 280285 Arg Leu Phe Leu Ala His Lys Leu Glu Ile Gly Ser Thr Ile Ser Ala 290295 300 Lys Ile Asn Ile Gly Pro Pro Gln Ala Ala Asp Ile Ala Pro Ala Lys305 310 315 320 Leu Gln Leu Val Tyr Tyr Gly Arg Gly Gly Arg Gly Asn TyrPhe Leu 325 330 335 Arg Val Ala Asp Asp Val Ser Leu Gly Gly Lys Leu LeuThr Asn Cys 340 345 350 Tyr Leu Thr Ser Asp Asp Gly Gln Ser Asn Asn IleSer Gly Pro Tyr 355 360 365 Cys Leu Met Ile Asn Arg Gly Thr Gly Ser MetPro Ser Gly Thr His 370 375 380 Leu Pro Val Gln Ile Glu Arg Val Thr AspThr Ser Ile Arg Ile Phe 385 390 395 400 Val Pro Asp His Gly Tyr Leu GlyLeu Gly Glu Ser Leu Ala Ser Asn 405 410 415 Trp Asn Glu Pro Leu Ala LeuAsn Leu Gly Leu Asp Glu Ala Leu Thr 420 425 430 Phe Thr Leu Arg Lys LysGlu Thr Gly Asn Asp Thr Ile Ser Ile Ile 435 440 445 Asp Met Leu Pro ProVal Ala Asn Thr Thr Pro Ser Pro Pro Thr Arg 450 455 460 Glu Thr Leu SerLeu Thr Pro Asn Ser Phe Arg Leu Leu Val Asn Pro 465 470 475 480 Glu ProThr Ala Glu Asp Ile Ala Lys His Tyr Asn Val Thr Thr Val 485 490 495 ThrArg Ala Pro Ala Asp Leu Ala Ser Ala Leu Asn Val Val Asp Asp 500 505 510Phe Cys Leu Lys Thr Gly Leu Ser Phe Asn Glu Leu Leu Asp Leu Thr 515 520525 Met Gln Lys Asp Tyr Gln Ser Lys Ser Ser Glu Tyr Lys Ser Arg Phe 530535 540 Val Lys Phe Gly Gly Gly Glu Asn Val Pro Val Ser Ser Tyr Gly Ala545 550 555 560 Ala Phe Leu Thr Gly Ala Glu Asp Thr Pro Leu Trp Val LysGln Tyr 565 570 575 Asn Ser Val Gly Thr Ala Thr Ser Thr Pro Val Leu AsnPhe Thr Pro 580 585 590 Asp Asn Val Val Ala Leu Ala Gly Arg Ala Glu LysLeu Val Arg Leu 595 600 605 Met Arg Ser Thr Gly Leu Ser Phe Glu Gln LeuAsp Trp Leu Ile Ala 610 615 620 Asn Ala Ser Arg Ala Val Ile Glu His GlyGly Glu Leu Phe Leu Asp 625 630 635 640 Lys Pro Val Leu Glu Ala Val AlaGlu Phe Thr Arg Leu Asn Lys Arg 645 650 655 Tyr Gly Val Thr Ser Asp MetPhe Ala Ala Phe Ile Gly Glu Val Asn 660 665 670 Thr Tyr Thr Glu Ala GlyLys Asp Ser Phe Tyr Gln Ala Ser Phe Ser 675 680 685 Thr Ala Asp His SerAla Thr Leu Pro Leu Gly Ala Ser Leu Gln Leu 690 695 700 Glu Val Ser LysGln Asp Arg Tyr Glu Ala Ile Cys Cys Gly Ala Met 705 710 715 720 Gly ValThr Ala Asp Glu Phe Ser Arg Ile Gly Lys Tyr Cys Phe Gly 725 730 735 AspLys Ala Gln Gln Ile Thr Ala Asn Glu Thr Thr Val Ala Gln Leu 740 745 750Tyr Arg Leu Gly Arg Ile Pro His Met Leu Gly Leu Arg Phe Thr Glu 755 760765 Ala Glu Leu Leu Trp Lys Leu Met Ala Gly Gly Glu Asp Thr Leu Leu 770775 780 Arg Thr Ile Gly Ala Asn Pro Arg Ser Leu Glu Ala Leu Glu Ile Ile785 790 795 800 Arg Arg Thr Glu Val Leu Leu Asp Trp Met Asp Ala His GlnLeu Asp 805 810 815 Val Val Ser Leu Gln Ala Met Val Thr Asn Arg Tyr SerGly Thr Ala 820 825 830 Thr Pro Glu Leu Tyr Asn Phe Leu Ala Gln Val HisGln Ser Ala Ser 835 840 845 Ser Ala Ala Asn Val Ala Arg Ala Asp Gly GlnAsp Thr Leu Pro Ala 850 855 860 Asp Lys Leu Leu Arg Ala Leu Ala Ala GlyPhe Lys Leu Lys Ala Asn 865 870 875 880 Val Met Ala Arg Val Ile Asp TrpMet Asp Lys Thr Asn Lys Ala Phe 885 890 895 Thr Leu Arg Ala Phe Trp AspLys Leu Gln Ala Tyr Phe Ser Ala Asp 900 905 910 His Glu Glu Glu Leu ThrAla Leu Glu Gly Glu Ala Ala Met Leu Gln 915 920 925 Trp Cys Gln Gln IleSer Gln Tyr Ala Leu Ile Val Arg Trp Cys Gly 930 935 940 Leu Ser Glu GlnAsp Leu Ala Leu Leu Thr Gly Asn Pro Glu Gln Leu 945 950 955 960 Leu AspGly Gln His Thr Val Pro Val Pro Ser Leu His Leu Leu Leu 965 970 975 ValLeu Thr Arg Leu Lys Glu Trp Gln Gln Arg Val Gln Val Ser Ser 980 985 990Glu Glu Ala Met Arg Tyr Phe Ala Gln Ala Asp Ser Pro Thr Val Thr 995 10001005 Arg Asp Asp Ala Val Asn Leu Leu Ala Arg Ile His Gly Trp Asn 10101015 1020 Glu Ala Asp Thr Val Ser Met Asn Asp Tyr Leu Leu Gly Glu Asn1025 1030 1035 Glu Tyr Pro Lys Asn Phe Asp Gln Ile Phe Ala Leu Glu SerTrp 1040 1045 1050 Val Asn Leu Gly Arg Gln Leu Asn Val Gly Ser Arg ThrLeu Gly 1055 1060 1065 Glu Leu Val Asp Met Ala Glu Glu Asp Lys Thr AlaGlu Asn Met 1070 1075 1080 Asp Leu Ile Thr Ser Val Ala His Ser Leu MetAla Ala Ala Lys 1085 1090 1095 Ala 8 3627 DNA Paenibacillus strain IDAS1529 8 atgaccaagg aaggtgataa gcatatgtct acttcaaccc tgttgcaatc gattaaagaa60 gcccgccggg atgcgctggt caaccattat attgctaatc aggttccgac agcgcttgcg 120gacaagatta cggacgcgga cagcctgtat gagtacttgc tgctggatac caagatcagt 180gaactcgtaa aaacatcgcc gatagcggag gccatcagca gcgtgcagtt atacatgaac 240cgctgcgtcg aaggctatga aggcaagttg actccggaaa gtaatactca ttttggccca 300ggtaaatttc tatataactg ggatacgtac aacaaacgtt tttccacctg ggcaggaaaa 360gaacgcttga aatattatgc aggcagctat attgagccgt ccttgcgcta caacaaaacc 420gatccattcc tgaacctgga acagagcatc agccagggaa gaattactga tgataccgta 480aagaacgcgc tgcaacacta cctgactgaa tatgaagtgt tggcggatct ggattatatc 540agcgttaata aaggcggcga cgaaagtgtt ttactctttg ttggacgcac caaaaccgta 600ccgtatgaat actactggcg ccgtttgctt ttaaaaaggg acaataataa taagctagta 660ccagcagtct ggtctcagtg gaaaaaaatc agtgccaata tcggtgaagc ggttgatagt 720tatgtggtgc ctcggtggca taaaaaccgg ctacatgtgc aatggtgttc tatagagaaa 780agtgaaaatg atgccggtga acccattgag aaacgatatt tgaatgactg gttcatggat 840agttccggag tctggtcttc atttcgaaag attccggttg tggaaaagag tttcgaatat 900ttggacggaa gcctcgatcc ccgatttgtc gctcttgtta gaaatcaaat attaattgat 960gagccagaaa tattcagaat tacagtatca gcccctaatc cgatagatgc aaatggaaga 1020gtagaggtac attttgaaga aaactatgca aacagatata atattaccat taaatatggg 1080acaacgagtc ttgctattcc tgcagggcag gtagggcatc caaatatctc tattaatgaa 1140acattaaggg ttgaattcgg caccaggccg gattggtatt atactttcag atatttagga 1200aatacaatcc aaaactcata cggttcaatt gtcaataatc aattttcacc tccatcagga 1260agcaatatta aaggtcctat cgaccttacc ctgaaaaata acatcgacct gtcggccttg 1320ttggatgaga gccttgacgc actgttcgac tataccattc agggcgataa ccaattgggc 1380ggcttagctg cctttaacgg gccttacgga ctttacttgt gggaaatctt cttccatgtt 1440ccttttttaa tggcggttcg cttccacacc gagcagcggt atgagttggc ggaacgttgg 1500tttaaattca tcttcaacag cgcaggatac cgtgatgatt acggcagtct gctgacggat 1560gacaaaggca acgtgcgtta ctggaacgtg ataccgctgc aagaggacac ggagtgggat 1620gacacgttgt ccctggcaac gaccgacccg gacgagattg cgatggccga cccgatgcaa 1680tacaagctgg ctatatttat tcacaccatg gacttcctga tcagccgcgg cgatagcttg 1740taccggatgc tggagcggga taccctggcc gaagccaaga tgtattacat tcaggccagc 1800caactgcttg ggccccgccc cgacatccgg ctcaatcaca gttggcctaa tccgaccttg 1860caaagcgaag cggacgcggt aaccgccgtg ccgacgcgaa gcgattcgcc ggcagcgcca 1920attttggcct tgcgagcgct tctgacaggc gaaaacggtc atttcctgcc gccttataat 1980gatgaactgt tcgctttctg ggacaaaatc gatctgcgtt tatacaattt gcgccacaat 2040ttgagtctgg acggtcagcc gcttcatttg ccgctctttg ccgaaccggt caatccgcgt 2100gaattgcagg ttcagcatgg cccgggcgat ggcttggggg gaagcgcggg ttccgcccaa 2160agccgtcaga gtgtctatcg ttttcctctg gtcatcgata aggcgcgcaa tgcggccaac 2220agtgtcatcc aattcggcaa tgccctggaa aacgcactga ccaagcaaga cagcgaagca 2280atgaccatgc tgttgcagtc ccagcagcag attgtcctgc agcaaacccg cgatattcag 2340gagaagaacc tggccgcgct gcaagcaagt ctggaagcaa cgatgacagc gaaagcgggg 2400gcggagtccc ggaagaccca ttttgccggc ttggcggaca actggatgtc ggacaatgaa 2460accgcctcac tcgcactgcg taccaccgcg ggaatcatca ataccagctc aaccgtgccg 2520atcgccatca ccggcggctt ggatatggct ccgaacattt ttggtttcgc agttggaggt 2580tcccgctggg gagcagccag cgcggctgta gcccaaggat tgcaaatcgc cgccggcgta 2640atggaacaga cggccaatat tatcgatatt agcgaaagct accgccggcg ccgggaggat 2700tggctgctgc agcgggatgt tgccgaaaat gaagcggcgc agttggattc gcagattgcg 2760gccctgcggg aacagatgga tatggcgcgc aagcaacttg cgctggcgga gacggaacag 2820gcgcacgcgc aagcggtcta cgagctgcaa agcacccgct ttacgaatca agctttgtat 2880aactggatgg ctggacgtct gtcgtctcta tactatcaaa tgtatgacgc cgcattgccg 2940ctctgcttga tggcgaagca ggctttagag aaagaaatcg gttcggataa aacggtcgga 3000gtcttgtccc tcccggcctg gaatgatcta tatcagggat tattggcggg cgaggcgctg 3060ctgctcgagc ttcagaagct ggagaatctg tggctggagg aagacaagcg cggaatggaa 3120gccgtaaaaa cagtctctct ggatactctt ctccgcaaaa caaatccgaa ctccgggttt 3180gcggatctcg tcaaggaggc actggacgaa aacggaaaga cgcctgaccc ggtgagcgga 3240gtcggcgtac agctgcaaaa caatattttc agcgcaaccc ttgacctctc cgttcttggc 3300ctggatcgct cttacaatca ggcggaaaag tcccgcagga tcaaaaatat gtcggttacc 3360ttacctgcgc tattggggcc ttaccaggat atagaggcaa ccttatcgct aggcggcgag 3420accgttgcgc tgtcccatgg cgtggatgac agcggcttgt tcatcactga tctcaacgac 3480agccggttcc tgcctttcga gggcatggat ccgttatccg gcacactcgt cctgtcgata 3540ttccatgccg ggcaagacgg cgaccagcgc ctcctgctgg aaagtctcaa tgacgtcatc 3600ttccacattc gatatgttat gaaatag 3627 9 1208 PRT Paenibacillus strain IDAS1529 9 Met Thr Lys Glu Gly Asp Lys His Met Ser Thr Ser Thr Leu Leu Gln 15 10 15 Ser Ile Lys Glu Ala Arg Arg Asp Ala Leu Val Asn His Tyr Ile Ala20 25 30 Asn Gln Val Pro Thr Ala Leu Ala Asp Lys Ile Thr Asp Ala Asp Ser35 40 45 Leu Tyr Glu Tyr Leu Leu Leu Asp Thr Lys Ile Ser Glu Leu Val Lys50 55 60 Thr Ser Pro Ile Ala Glu Ala Ile Ser Ser Val Gln Leu Tyr Met Asn65 70 75 80 Arg Cys Val Glu Gly Tyr Glu Gly Lys Leu Thr Pro Glu Ser AsnThr 85 90 95 His Phe Gly Pro Gly Lys Phe Leu Tyr Asn Trp Asp Thr Tyr AsnLys 100 105 110 Arg Phe Ser Thr Trp Ala Gly Lys Glu Arg Leu Lys Tyr TyrAla Gly 115 120 125 Ser Tyr Ile Glu Pro Ser Leu Arg Tyr Asn Lys Thr AspPro Phe Leu 130 135 140 Asn Leu Glu Gln Ser Ile Ser Gln Gly Arg Ile ThrAsp Asp Thr Val 145 150 155 160 Lys Asn Ala Leu Gln His Tyr Leu Thr GluTyr Glu Val Leu Ala Asp 165 170 175 Leu Asp Tyr Ile Ser Val Asn Lys GlyGly Asp Glu Ser Val Leu Leu 180 185 190 Phe Val Gly Arg Thr Lys Thr ValPro Tyr Glu Tyr Tyr Trp Arg Arg 195 200 205 Leu Leu Leu Lys Arg Asp AsnAsn Asn Lys Leu Val Pro Ala Val Trp 210 215 220 Ser Gln Trp Lys Lys IleSer Ala Asn Ile Gly Glu Ala Val Asp Ser 225 230 235 240 Tyr Val Val ProArg Trp His Lys Asn Arg Leu His Val Gln Trp Cys 245 250 255 Ser Ile GluLys Ser Glu Asn Asp Ala Gly Glu Pro Ile Glu Lys Arg 260 265 270 Tyr LeuAsn Asp Trp Phe Met Asp Ser Ser Gly Val Trp Ser Ser Phe 275 280 285 ArgLys Ile Pro Val Val Glu Lys Ser Phe Glu Tyr Leu Asp Gly Ser 290 295 300Leu Asp Pro Arg Phe Val Ala Leu Val Arg Asn Gln Ile Leu Ile Asp 305 310315 320 Glu Pro Glu Ile Phe Arg Ile Thr Val Ser Ala Pro Asn Pro Ile Asp325 330 335 Ala Asn Gly Arg Val Glu Val His Phe Glu Glu Asn Tyr Ala AsnArg 340 345 350 Tyr Asn Ile Thr Ile Lys Tyr Gly Thr Thr Ser Leu Ala IlePro Ala 355 360 365 Gly Gln Val Gly His Pro Asn Ile Ser Ile Asn Glu ThrLeu Arg Val 370 375 380 Glu Phe Gly Thr Arg Pro Asp Trp Tyr Tyr Thr PheArg Tyr Leu Gly 385 390 395 400 Asn Thr Ile Gln Asn Ser Tyr Gly Ser IleVal Asn Asn Gln Phe Ser 405 410 415 Pro Pro Ser Gly Ser Asn Ile Lys GlyPro Ile Asp Leu Thr Leu Lys 420 425 430 Asn Asn Ile Asp Leu Ser Ala LeuLeu Asp Glu Ser Leu Asp Ala Leu 435 440 445 Phe Asp Tyr Thr Ile Gln GlyAsp Asn Gln Leu Gly Gly Leu Ala Ala 450 455 460 Phe Asn Gly Pro Tyr GlyLeu Tyr Leu Trp Glu Ile Phe Phe His Val 465 470 475 480 Pro Phe Leu MetAla Val Arg Phe His Thr Glu Gln Arg Tyr Glu Leu 485 490 495 Ala Glu ArgTrp Phe Lys Phe Ile Phe Asn Ser Ala Gly Tyr Arg Asp 500 505 510 Asp TyrGly Ser Leu Leu Thr Asp Asp Lys Gly Asn Val Arg Tyr Trp 515 520 525 AsnVal Ile Pro Leu Gln Glu Asp Thr Glu Trp Asp Asp Thr Leu Ser 530 535 540Leu Ala Thr Thr Asp Pro Asp Glu Ile Ala Met Ala Asp Pro Met Gln 545 550555 560 Tyr Lys Leu Ala Ile Phe Ile His Thr Met Asp Phe Leu Ile Ser Arg565 570 575 Gly Asp Ser Leu Tyr Arg Met Leu Glu Arg Asp Thr Leu Ala GluAla 580 585 590 Lys Met Tyr Tyr Ile Gln Ala Ser Gln Leu Leu Gly Pro ArgPro Asp 595 600 605 Ile Arg Leu Asn His Ser Trp Pro Asn Pro Thr Leu GlnSer Glu Ala 610 615 620 Asp Ala Val Thr Ala Val Pro Thr Arg Ser Asp SerPro Ala Ala Pro 625 630 635 640 Ile Leu Ala Leu Arg Ala Leu Leu Thr GlyGlu Asn Gly His Phe Leu 645 650 655 Pro Pro Tyr Asn Asp Glu Leu Phe AlaPhe Trp Asp Lys Ile Asp Leu 660 665 670 Arg Leu Tyr Asn Leu Arg His AsnLeu Ser Leu Asp Gly Gln Pro Leu 675 680 685 His Leu Pro Leu Phe Ala GluPro Val Asn Pro Arg Glu Leu Gln Val 690 695 700 Gln His Gly Pro Gly AspGly Leu Gly Gly Ser Ala Gly Ser Ala Gln 705 710 715 720 Ser Arg Gln SerVal Tyr Arg Phe Pro Leu Val Ile Asp Lys Ala Arg 725 730 735 Asn Ala AlaAsn Ser Val Ile Gln Phe Gly Asn Ala Leu Glu Asn Ala 740 745 750 Leu ThrLys Gln Asp Ser Glu Ala Met Thr Met Leu Leu Gln Ser Gln 755 760 765 GlnGln Ile Val Leu Gln Gln Thr Arg Asp Ile Gln Glu Lys Asn Leu 770 775 780Ala Ala Leu Gln Ala Ser Leu Glu Ala Thr Met Thr Ala Lys Ala Gly 785 790795 800 Ala Glu Ser Arg Lys Thr His Phe Ala Gly Leu Ala Asp Asn Trp Met805 810 815 Ser Asp Asn Glu Thr Ala Ser Leu Ala Leu Arg Thr Thr Ala GlyIle 820 825 830 Ile Asn Thr Ser Ser Thr Val Pro Ile Ala Ile Thr Gly GlyLeu Asp 835 840 845 Met Ala Pro Asn Ile Phe Gly Phe Ala Val Gly Gly SerArg Trp Gly 850 855 860 Ala Ala Ser Ala Ala Val Ala Gln Gly Leu Gln IleAla Ala Gly Val 865 870 875 880 Met Glu Gln Thr Ala Asn Ile Ile Asp IleSer Glu Ser Tyr Arg Arg 885 890 895 Arg Arg Glu Asp Trp Leu Leu Gln ArgAsp Val Ala Glu Asn Glu Ala 900 905 910 Ala Gln Leu Asp Ser Gln Ile AlaAla Leu Arg Glu Gln Met Asp Met 915 920 925 Ala Arg Lys Gln Leu Ala LeuAla Glu Thr Glu Gln Ala His Ala Gln 930 935 940 Ala Val Tyr Glu Leu GlnSer Thr Arg Phe Thr Asn Gln Ala Leu Tyr 945 950 955 960 Asn Trp Met AlaGly Arg Leu Ser Ser Leu Tyr Tyr Gln Met Tyr Asp 965 970 975 Ala Ala LeuPro Leu Cys Leu Met Ala Lys Gln Ala Leu Glu Lys Glu 980 985 990 Ile GlySer Asp Lys Thr Val Gly Val Leu Ser Leu Pro Ala Trp Asn 995 1000 1005Asp Leu Tyr Gln Gly Leu Leu Ala Gly Glu Ala Leu Leu Leu Glu 1010 10151020 Leu Gln Lys Leu Glu Asn Leu Trp Leu Glu Glu Asp Lys Arg Gly 10251030 1035 Met Glu Ala Val Lys Thr Val Ser Leu Asp Thr Leu Leu Arg Lys1040 1045 1050 Thr Asn Pro Asn Ser Gly Phe Ala Asp Leu Val Lys Glu AlaLeu 1055 1060 1065 Asp Glu Asn Gly Lys Thr Pro Asp Pro Val Ser Gly ValGly Val 1070 1075 1080 Gln Leu Gln Asn Asn Ile Phe Ser Ala Thr Leu AspLeu Ser Val 1085 1090 1095 Leu Gly Leu Asp Arg Ser Tyr Asn Gln Ala GluLys Ser Arg Arg 1100 1105 1110 Ile Lys Asn Met Ser Val Thr Leu Pro AlaLeu Leu Gly Pro Tyr 1115 1120 1125 Gln Asp Ile Glu Ala Thr Leu Ser LeuGly Gly Glu Thr Val Ala 1130 1135 1140 Leu Ser His Gly Val Asp Asp SerGly Leu Phe Ile Thr Asp Leu 1145 1150 1155 Asn Asp Ser Arg Phe Leu ProPhe Glu Gly Met Asp Pro Leu Ser 1160 1165 1170 Gly Thr Leu Val Leu SerIle Phe His Ala Gly Gln Asp Gly Asp 1175 1180 1185 Gln Arg Leu Leu LeuGlu Ser Leu Asn Asp Val Ile Phe His Ile 1190 1195 1200 Arg Tyr Val MetLys 1205 10 4335 DNA Paenibacillus strain IDAS 1529 10 atgccacaatctagcaatgc cgatatcaag ctattgtcgc catcgctgcc aaagggcggc 60 ggttccatgaagggaatcga agaaaacatc gcggctcccg gctccgacgg catggcacgt 120 tgtaatgtgccgctgccggt aacctccggc cgctatatta ctcctgatat aagcctgtcc 180 tatgcgagcggccacggcaa cggcgcttat ggaatgggct ggacgatggg agtgatgagc 240 attagccggagaacaagccg agggaccccc agttatacat ccgaagacca gttccttggt 300 ccggatggggaggtgcttgt tccggaaagc aacgaacaag gggagatcat tacccgccac 360 accgatacggcccaagggat accgttaggc gagacgttta cggttacacg ctattttccc 420 cggatcgagagcgcttttca tttgctggaa tactgggaag cgcaagcagg aagcgcaaca 480 gcgtcgttttggcttattca ctctgccgat ggagtgctgc actgtctggg taaaactgct 540 caggcgaggatagccgcccc tgacgattcc gccaagatcg cagaatggct agtggaggag 600 tccgtctcccccttcggaga gcatatttat taccaataca aagaagaaga caatcaaggc 660 gtgaatctggaggaagacaa tcatcaatat ggggcgaacc gctatctgaa atcgattcgc 720 tatggaaataaggttgcctc tccttctctc tatgtctgga agggggaaat tccggcagac 780 ggccaatggctgtattccgt tatcctggat tatggcgaga acgatacctc agcggatgtt 840 cctcccctatacacgcccca aggggagtgg ctggtgcgcc cggaccgttt ttcccgctat 900 gactacggatttgaggtccg gacttgccgc ttgtgccgcc aggtcttgat gttccacgtc 960 tttaaggagcttggcgggga gccggcgctg gtgtggcgga tgcagttgga atacgacgag 1020 aacccggcggcgtccatgct gagcgcggtc cggcaattgg cttatgaagc agatggggcc 1080 attcgaagcttgccgccgct ggaattcgat tatactccat ttggcatcga gacaacggcc 1140 gattggcagccttttctgcc tgtgcctgaa tgggcggatg aagaacatta tcagttggtc 1200 gatttgtacggagaaggcat accgggctta ttatatcaga acaatgacca ctggcattat 1260 cgttcgcccgcccggggcga cacaccggac gggatcgcct ataacagctg gcggccgctt 1320 cctcatatccccgtgaactc ccggaacggg atgctgatgg atctgaatgg agacgggtat 1380 ctggaatggttgcttgcgga acccggggtt gcggggcgct atagcatgaa cccggataag 1440 agctggtccggttttgtgcc gctccaggca ctgccaacgg aattcttcca tccgcaggca 1500 cagcttgccaatgttaccgg atcgggttta accgacttgg ttatgatcgg tccgaagagc 1560 gtccggttttatgccggaga agaagcgggc ttcaagcgcg catgtgaagt gtggcagcaa 1620 gtgggcattactttgcctgt ggaacgcgtg gataaaaagg aactggtggc attcagcgat 1680 atgctgggatcgggtcagtc tcatctggtg cgcatccggc atgatggcgt tacatgctgg 1740 cctaatctggggaacggcgt gttcggggcg ccgttggccc ttcacgggtt tacggcatcg 1800 gagcgggaattcaatccgga acgtgtatat cttgtggacc ttgatggatc cggcgcttcc 1860 gatatcatttatgcttctcg tgacgctcta ctcatttacc gaaatctttc cggcaatggc 1920 tttgctgatccggtgcgggt tccgctgcct gacggcgtgc ggtttgataa tctgtgccgg 1980 ctgctgcctgccgatatccg cgggttaggt gtggccagtc tggtgctgca tgtaccttac 2040 atggccccccgcagttggaa attagatttc tttgcggcga agccgtattt attgcaaacg 2100 gtcagcaacaatcttggagc ttccagctcg ttttggtacc gaagctccac ccagtattgg 2160 ctggatgagaaacaggcggc ctcatcggct gtctccgctt tgcccttccc gataaacgtg 2220 gtatcggatatgcacacggt ggacgaaatc agcggccgca ccaggactca gaagtatact 2280 taccgccatggcgtgtatga ccggaccgaa aaggaatttg ccggattcgg ccgcattgac 2340 acatgggaagaggagcggga ttccgaagga accctgagcg tcagcactcc gcccgtgctg 2400 acgcggacctggtatcatac cgggcaaaag caggatgagg agcgtgccgt gcagcaatat 2460 tggcaaggcgaccctgcggc ttttcaggtt aaacccgtcc ggcttactcg attcgatgcg 2520 gcagcggcccaggatctgcc gctagattct aataatgggc agcaagaata ctggctgtac 2580 cgatcattacaagggatgcc gctgcggact gagatttttg cgggagatgt tggcgggtcg 2640 cctccttatcaggtagagag cttccgttat caagtgcgct tggtgcagag catcgattcg 2700 gaatgtgttgccttgcccat gcagttggag cagcttacgt acaactatga gcaaatcgcc 2760 tctgatccgcagtgttcaca gcagatacag caatggttcg acgaatacgg cgtggcggca 2820 cagagtgtaacaatccaata tccgcgccgg gcacagccgg aggacaatcc gtaccctcgc 2880 acgctgccggataccagctg gagcagcagt tatgattcgc agcaaatgct gctgcggttg 2940 accaggcaaaggcaaaaagc gtaccacctt gcagatcctg aaggctggcg cttgaatatt 3000 ccccatcagacacgcctgga tgccttcatt tattctgctg acagcgtgcc cgccgaagga 3060 ataagcgccgagctgctgga ggtggacggc acgttacgat cttcggcgct ggaacaggct 3120 tatggcggccagtcagagat catctatgcg ggcgggggcg aaccggattt gcgagccctg 3180 gtccattacaccagaagcgc ggttcttgat gaagactgtt tacaagccta tgaaggcgta 3240 ctgagcgatagccaattgaa ctcgcttctt gcctcttccg gctatcaacg aagcgcaaga 3300 atattgggttcgggcgatga agtggatatt tttgtcgcgg aacaaggatt tacccgttat 3360 gcggatgaaccgaatttttt ccgtattctg gggcaacaat cctctctctt gtccggggaa 3420 caagtattaacatgggatga taatttctgt gcggttacat ccatcgaaga cgcgcttggc 3480 aatcaaattcagattgcata tgattaccgc tttgtggagg ccatccagat taccgatacg 3540 aataataatgtgaatcaggt cgccctggat gctctcggcc gggtcgtata cagccggacc 3600 tggggcacggaggaagggat aaagaccggc ttccgcccgg aggtggaatt cgcgacgccc 3660 gagacaatggagcaggcgct tgccctggca tctcccttgc cggttgcatc ctgctgtgta 3720 tatgatgcgcatagctggat gggaacgata actcttgcac aactgtcaga gcttgttcca 3780 gatagtgaaaagcaatggtc gttcttgata gacaatcgct tgattatgcc ggacggcaga 3840 atcagatcccgcggtcggga tccatggtcg cttcaccggc tattgccgcc tgctgtgggc 3900 gaattgctgagcgaggcgga ccgtaaaccg ccgcatacgg taattttggc agcagatcgt 3960 tacccggatgacccatccca gcaaattcag gcgagcatcg tgtttagcga tggctttggg 4020 cgtacgatacaaactgctaa aagagaagat acccgatggg cgattgcgga acgggtggac 4080 tatgacggaaccggagccgt aatccgcagc tttcagcctt tttatcttga cgactggaat 4140 tatgtgggcgaagaggctgt cagcagctct atgtacgcaa cgatctatta ttatgatgct 4200 ctggcacgacaattaaggat ggtcaacgct aaaggatatg agaggagaac tgctttttac 4260 ccatggtttacagtaaacga agatgaaaat gataccatgg actcatcatt atttgcttca 4320 ccgcctgcgcggtga 4335 11 1444 PRT Paenibacillus strain IDAS 1529 11 Met Pro Gln SerSer Asn Ala Asp Ile Lys Leu Leu Ser Pro Ser Leu 1 5 10 15 Pro Lys GlyGly Gly Ser Met Lys Gly Ile Glu Glu Asn Ile Ala Ala 20 25 30 Pro Gly SerAsp Gly Met Ala Arg Cys Asn Val Pro Leu Pro Val Thr 35 40 45 Ser Gly ArgTyr Ile Thr Pro Asp Ile Ser Leu Ser Tyr Ala Ser Gly 50 55 60 His Gly AsnGly Ala Tyr Gly Met Gly Trp Thr Met Gly Val Met Ser 65 70 75 80 Ile SerArg Arg Thr Ser Arg Gly Thr Pro Ser Tyr Thr Ser Glu Asp 85 90 95 Gln PheLeu Gly Pro Asp Gly Glu Val Leu Val Pro Glu Ser Asn Glu 100 105 110 GlnGly Glu Ile Ile Thr Arg His Thr Asp Thr Ala Gln Gly Ile Pro 115 120 125Leu Gly Glu Thr Phe Thr Val Thr Arg Tyr Phe Pro Arg Ile Glu Ser 130 135140 Ala Phe His Leu Leu Glu Tyr Trp Glu Ala Gln Ala Gly Ser Ala Thr 145150 155 160 Ala Ser Phe Trp Leu Ile His Ser Ala Asp Gly Val Leu His CysLeu 165 170 175 Gly Lys Thr Ala Gln Ala Arg Ile Ala Ala Pro Asp Asp SerAla Lys 180 185 190 Ile Ala Glu Trp Leu Val Glu Glu Ser Val Ser Pro PheGly Glu His 195 200 205 Ile Tyr Tyr Gln Tyr Lys Glu Glu Asp Asn Gln GlyVal Asn Leu Glu 210 215 220 Glu Asp Asn His Gln Tyr Gly Ala Asn Arg TyrLeu Lys Ser Ile Arg 225 230 235 240 Tyr Gly Asn Lys Val Ala Ser Pro SerLeu Tyr Val Trp Lys Gly Glu 245 250 255 Ile Pro Ala Asp Gly Gln Trp LeuTyr Ser Val Ile Leu Asp Tyr Gly 260 265 270 Glu Asn Asp Thr Ser Ala AspVal Pro Pro Leu Tyr Thr Pro Gln Gly 275 280 285 Glu Trp Leu Val Arg ProAsp Arg Phe Ser Arg Tyr Asp Tyr Gly Phe 290 295 300 Glu Val Arg Thr CysArg Leu Cys Arg Gln Val Leu Met Phe His Val 305 310 315 320 Phe Lys GluLeu Gly Gly Glu Pro Ala Leu Val Trp Arg Met Gln Leu 325 330 335 Glu TyrAsp Glu Asn Pro Ala Ala Ser Met Leu Ser Ala Val Arg Gln 340 345 350 LeuAla Tyr Glu Ala Asp Gly Ala Ile Arg Ser Leu Pro Pro Leu Glu 355 360 365Phe Asp Tyr Thr Pro Phe Gly Ile Glu Thr Thr Ala Asp Trp Gln Pro 370 375380 Phe Leu Pro Val Pro Glu Trp Ala Asp Glu Glu His Tyr Gln Leu Val 385390 395 400 Asp Leu Tyr Gly Glu Gly Ile Pro Gly Leu Leu Tyr Gln Asn AsnAsp 405 410 415 His Trp His Tyr Arg Ser Pro Ala Arg Gly Asp Thr Pro AspGly Ile 420 425 430 Ala Tyr Asn Ser Trp Arg Pro Leu Pro His Ile Pro ValAsn Ser Arg 435 440 445 Asn Gly Met Leu Met Asp Leu Asn Gly Asp Gly TyrLeu Glu Trp Leu 450 455 460 Leu Ala Glu Pro Gly Val Ala Gly Arg Tyr SerMet Asn Pro Asp Lys 465 470 475 480 Ser Trp Ser Gly Phe Val Pro Leu GlnAla Leu Pro Thr Glu Phe Phe 485 490 495 His Pro Gln Ala Gln Leu Ala AsnVal Thr Gly Ser Gly Leu Thr Asp 500 505 510 Leu Val Met Ile Gly Pro LysSer Val Arg Phe Tyr Ala Gly Glu Glu 515 520 525 Ala Gly Phe Lys Arg AlaCys Glu Val Trp Gln Gln Val Gly Ile Thr 530 535 540 Leu Pro Val Glu ArgVal Asp Lys Lys Glu Leu Val Ala Phe Ser Asp 545 550 555 560 Met Leu GlySer Gly Gln Ser His Leu Val Arg Ile Arg His Asp Gly 565 570 575 Val ThrCys Trp Pro Asn Leu Gly Asn Gly Val Phe Gly Ala Pro Leu 580 585 590 AlaLeu His Gly Phe Thr Ala Ser Glu Arg Glu Phe Asn Pro Glu Arg 595 600 605Val Tyr Leu Val Asp Leu Asp Gly Ser Gly Ala Ser Asp Ile Ile Tyr 610 615620 Ala Ser Arg Asp Ala Leu Leu Ile Tyr Arg Asn Leu Ser Gly Asn Gly 625630 635 640 Phe Ala Asp Pro Val Arg Val Pro Leu Pro Asp Gly Val Arg PheAsp 645 650 655 Asn Leu Cys Arg Leu Leu Pro Ala Asp Ile Arg Gly Leu GlyVal Ala 660 665 670 Ser Leu Val Leu His Val Pro Tyr Met Ala Pro Arg SerTrp Lys Leu 675 680 685 Asp Phe Phe Ala Ala Lys Pro Tyr Leu Leu Gln ThrVal Ser Asn Asn 690 695 700 Leu Gly Ala Ser Ser Ser Phe Trp Tyr Arg SerSer Thr Gln Tyr Trp 705 710 715 720 Leu Asp Glu Lys Gln Ala Ala Ser SerAla Val Ser Ala Leu Pro Phe 725 730 735 Pro Ile Asn Val Val Ser Asp MetHis Thr Val Asp Glu Ile Ser Gly 740 745 750 Arg Thr Arg Thr Gln Lys TyrThr Tyr Arg His Gly Val Tyr Asp Arg 755 760 765 Thr Glu Lys Glu Phe AlaGly Phe Gly Arg Ile Asp Thr Trp Glu Glu 770 775 780 Glu Arg Asp Ser GluGly Thr Leu Ser Val Ser Thr Pro Pro Val Leu 785 790 795 800 Thr Arg ThrTrp Tyr His Thr Gly Gln Lys Gln Asp Glu Glu Arg Ala 805 810 815 Val GlnGln Tyr Trp Gln Gly Asp Pro Ala Ala Phe Gln Val Lys Pro 820 825 830 ValArg Leu Thr Arg Phe Asp Ala Ala Ala Ala Gln Asp Leu Pro Leu 835 840 845Asp Ser Asn Asn Gly Gln Gln Glu Tyr Trp Leu Tyr Arg Ser Leu Gln 850 855860 Gly Met Pro Leu Arg Thr Glu Ile Phe Ala Gly Asp Val Gly Gly Ser 865870 875 880 Pro Pro Tyr Gln Val Glu Ser Phe Arg Tyr Gln Val Arg Leu ValGln 885 890 895 Ser Ile Asp Ser Glu Cys Val Ala Leu Pro Met Gln Leu GluGln Leu 900 905 910 Thr Tyr Asn Tyr Glu Gln Ile Ala Ser Asp Pro Gln CysSer Gln Gln 915 920 925 Ile Gln Gln Trp Phe Asp Glu Tyr Gly Val Ala AlaGln Ser Val Thr 930 935 940 Ile Gln Tyr Pro Arg Arg Ala Gln Pro Glu AspAsn Pro Tyr Pro Arg 945 950 955 960 Thr Leu Pro Asp Thr Ser Trp Ser SerSer Tyr Asp Ser Gln Gln Met 965 970 975 Leu Leu Arg Leu Thr Arg Gln ArgGln Lys Ala Tyr His Leu Ala Asp 980 985 990 Pro Glu Gly Trp Arg Leu AsnIle Pro His Gln Thr Arg Leu Asp Ala 995 1000 1005 Phe Ile Tyr Ser AlaAsp Ser Val Pro Ala Glu Gly Ile Ser Ala 1010 1015 1020 Glu Leu Leu GluVal Asp Gly Thr Leu Arg Ser Ser Ala Leu Glu 1025 1030 1035 Gln Ala TyrGly Gly Gln Ser Glu Ile Ile Tyr Ala Gly Gly Gly 1040 1045 1050 Glu ProAsp Leu Arg Ala Leu Val His Tyr Thr Arg Ser Ala Val 1055 1060 1065 LeuAsp Glu Asp Cys Leu Gln Ala Tyr Glu Gly Val Leu Ser Asp 1070 1075 1080Ser Gln Leu Asn Ser Leu Leu Ala Ser Ser Gly Tyr Gln Arg Ser 1085 10901095 Ala Arg Ile Leu Gly Ser Gly Asp Glu Val Asp Ile Phe Val Ala 11001105 1110 Glu Gln Gly Phe Thr Arg Tyr Ala Asp Glu Pro Asn Phe Phe Arg1115 1120 1125 Ile Leu Gly Gln Gln Ser Ser Leu Leu Ser Gly Glu Gln ValLeu 1130 1135 1140 Thr Trp Asp Asp Asn Phe Cys Ala Val Thr Ser Ile GluAsp Ala 1145 1150 1155 Leu Gly Asn Gln Ile Gln Ile Ala Tyr Asp Tyr ArgPhe Val Glu 1160 1165 1170 Ala Ile Gln Ile Thr Asp Thr Asn Asn Asn ValAsn Gln Val Ala 1175 1180 1185 Leu Asp Ala Leu Gly Arg Val Val Tyr SerArg Thr Trp Gly Thr 1190 1195 1200 Glu Glu Gly Ile Lys Thr Gly Phe ArgPro Glu Val Glu Phe Ala 1205 1210 1215 Thr Pro Glu Thr Met Glu Gln AlaLeu Ala Leu Ala Ser Pro Leu 1220 1225 1230 Pro Val Ala Ser Cys Cys ValTyr Asp Ala His Ser Trp Met Gly 1235 1240 1245 Thr Ile Thr Leu Ala GlnLeu Ser Glu Leu Val Pro Asp Ser Glu 1250 1255 1260 Lys Gln Trp Ser PheLeu Ile Asp Asn Arg Leu Ile Met Pro Asp 1265 1270 1275 Gly Arg Ile ArgSer Arg Gly Arg Asp Pro Trp Ser Leu His Arg 1280 1285 1290 Leu Leu ProPro Ala Val Gly Glu Leu Leu Ser Glu Ala Asp Arg 1295 1300 1305 Lys ProPro His Thr Val Ile Leu Ala Ala Asp Arg Tyr Pro Asp 1310 1315 1320 AspPro Ser Gln Gln Ile Gln Ala Ser Ile Val Phe Ser Asp Gly 1325 1330 1335Phe Gly Arg Thr Ile Gln Thr Ala Lys Arg Glu Asp Thr Arg Trp 1340 13451350 Ala Ile Ala Glu Arg Val Asp Tyr Asp Gly Thr Gly Ala Val Ile 13551360 1365 Arg Ser Phe Gln Pro Phe Tyr Leu Asp Asp Trp Asn Tyr Val Gly1370 1375 1380 Glu Glu Ala Val Ser Ser Ser Met Tyr Ala Thr Ile Tyr TyrTyr 1385 1390 1395 Asp Ala Leu Ala Arg Gln Leu Arg Met Val Asn Ala LysGly Tyr 1400 1405 1410 Glu Arg Arg Thr Ala Phe Tyr Pro Trp Phe Thr ValAsn Glu Asp 1415 1420 1425 Glu Asn Asp Thr Met Asp Ser Ser Leu Phe AlaSer Pro Pro Ala 1430 1435 1440 Arg 12 2793 DNA Paenibacillus strain IDAS1529 12 atgaacacaa cgtccatata taggggcacg cctacgattt cagttgtggataaccggaac 60 ttggagattc gcattcttca gtataaccgt atcgcggctg aagatccggcagatgagtgt 120 atcctgcgga acacgtatac gccgttaagc tatcttggca gcagcatggatccccgtttg 180 ttctcgcaat atcaggatga tcgcggaaca ccgccgaata tacgaaccatggcttccctg 240 agaggcgaag cgctgtgttc ggaaagtgtg gatgccggcc gcaaggcggagctttttgat 300 atcgaggggc ggcccgtctg gcttatcgat gccaacggca cagagacgactctcgaatat 360 gatgtcttag gcaggccaac agccgtattc gagcaacagg aaggtacggactccccccag 420 tgcagggagc ggtttattta tggtgagaag gaggcggatg cccaggccaacaatttgcgc 480 ggacaactgg ttcgccacta cgataccgcg ggccggatac agaccgacagcatctccttg 540 gctggactgc cgttgcgcca aagccgtcaa ctgctgaaaa attgggatgaacctggcgac 600 tggagtatgg atgaggaaag cgcctgggcc tcgttgctgg ctgccgaagcttatgatacg 660 agctggcggt atgacgcgca ggacagggtg ctcgcccaaa ccgacgccaaagggaatctc 720 cagcaactga cttacaatga cgccggccag ccgcaggcgg tcagcctcaagctgcaaggc 780 caagcggagc aacggatttg gaaccggatc gagtacaacg cggcgggtcaagtggatctc 840 gccgaagccg ggaatggaat cgtaacggaa tatacttacg aggaaagcacgcagcggtta 900 atccgaaaaa aagattcccg cggactgtcc tccggggaaa gagaagtgctgcaggattat 960 cgttatgaat atgatccggt aggcaatatc ctttctattt acaatgaagcggagccggtt 1020 cgttatttcc gcaatcaggc cgttgctccg aaaaggcaat atgcctacgatgccttgtat 1080 cagcttgtat ctagttcggg gcgggaatcc gacgcgcttc ggcagcagacgtcgcttcct 1140 cccttgatca cgcctatccc tctggacgat agccaatacg tcaattacgctgaaaaatac 1200 agctatgatc aggcgggcaa tttaatcaag cttagccata acggggcaagtcaatataca 1260 acgaatgtgt atgtggacaa aagctcaaac cgggggattt ggcggcaaggggaagacatc 1320 ccggatatcg cggcttcctt tgacagagca ggcaatcaac aagctttattcccggggaga 1380 ccgttggaat gggatacacg caatcaatta agccgtgtcc atatggtcgtgcgcgaaggc 1440 ggagacaacg actgggaagg ctatctctat gacagctcgg gaatgcgtatcgtaaaacga 1500 tctacccgca aaacacagac aacgacgcaa acggatacga ccctctatttgccgggcctg 1560 gagctgcgaa tccgccagac cggggaccgg gtcacggaag cattgcaggtcattaccgtg 1620 gatgagggag cgggacaagt gagggtgctg cactgggagg atggaaccgagccgggcggc 1680 atcgccaatg atcagtaccg gtacagcctg aacgatcatc ttacctcctctttattggaa 1740 gttgacgggc aaggtcagat cattagtaag gaagaatttt atccctatggcggcacagcc 1800 ctgtggacag cccggtcaga ggtagaggca agctacaaga ccatccgctattcaggcaaa 1860 gagcgggatg ccacaggcct gtattattac ggacaccgct actatatgccatggttgggt 1920 cgctggctga atccggaccc ggccggaatg gtagatggac taaacctgtaccgtatggtc 1980 aggaacaatc ctataggact gatggatccg aatgggaatg cgccaatcaacgtggcggat 2040 tatagcttcg tgcatggtga tttagtttat ggtcttagta aggaaagaggaagatatcta 2100 aagctattta atccaaactt taatatggaa aaatcagact ctcctgctatggttatagat 2160 caatataata ataatgttgc attgagtata actaaccaat ataaagtagaagaattgatg 2220 aaatttcaaa aagacccaca aaaagccgca cggaaaataa aggttccagaagggaatcgt 2280 ttatcgagga acgaaaatta tcctttgtgg cacgattata ttaacattggagaagctaaa 2340 gctgcattta aggcctctca tattttccaa gaagtgaagg ggaattatgggaaagattat 2400 tatcataaat tattattaga cagaatgata gaatcgccgt tgctgtggaaacgaggcagc 2460 aaactcgggc tagaaatcgc cgctaccaat cagagaacaa aaatacactttgttcttgac 2520 aatttaaata tcgagcaggt ggttacgaaa gagggtagcg gcggtcagtcaatcacagct 2580 tcggagctcc gttatattta tcgaaatcgc gaaagattga acgggcgtgtcattttctat 2640 agaaataatg aaaggctaga tcaggctcca tggcaagaaa atccggacttatggagcaaa 2700 tatcaaccgg gtcttagaca aagcagcagt tcaagagtca aagaacgagggattgggaac 2760 tttttccgcc ggttttcaat gaagagaaag tag 2793 13 930 PRTPaenibacillus strain IDAS 1529 13 Met Asn Thr Thr Ser Ile Tyr Arg GlyThr Pro Thr Ile Ser Val Val 1 5 10 15 Asp Asn Arg Asn Leu Glu Ile ArgIle Leu Gln Tyr Asn Arg Ile Ala 20 25 30 Ala Glu Asp Pro Ala Asp Glu CysIle Leu Arg Asn Thr Tyr Thr Pro 35 40 45 Leu Ser Tyr Leu Gly Ser Ser MetAsp Pro Arg Leu Phe Ser Gln Tyr 50 55 60 Gln Asp Asp Arg Gly Thr Pro ProAsn Ile Arg Thr Met Ala Ser Leu 65 70 75 80 Arg Gly Glu Ala Leu Cys SerGlu Ser Val Asp Ala Gly Arg Lys Ala 85 90 95 Glu Leu Phe Asp Ile Glu GlyArg Pro Val Trp Leu Ile Asp Ala Asn 100 105 110 Gly Thr Glu Thr Thr LeuGlu Tyr Asp Val Leu Gly Arg Pro Thr Ala 115 120 125 Val Phe Glu Gln GlnGlu Gly Thr Asp Ser Pro Gln Cys Arg Glu Arg 130 135 140 Phe Ile Tyr GlyGlu Lys Glu Ala Asp Ala Gln Ala Asn Asn Leu Arg 145 150 155 160 Gly GlnLeu Val Arg His Tyr Asp Thr Ala Gly Arg Ile Gln Thr Asp 165 170 175 SerIle Ser Leu Ala Gly Leu Pro Leu Arg Gln Ser Arg Gln Leu Leu 180 185 190Lys Asn Trp Asp Glu Pro Gly Asp Trp Ser Met Asp Glu Glu Ser Ala 195 200205 Trp Ala Ser Leu Leu Ala Ala Glu Ala Tyr Asp Thr Ser Trp Arg Tyr 210215 220 Asp Ala Gln Asp Arg Val Leu Ala Gln Thr Asp Ala Lys Gly Asn Leu225 230 235 240 Gln Gln Leu Thr Tyr Asn Asp Ala Gly Gln Pro Gln Ala ValSer Leu 245 250 255 Lys Leu Gln Gly Gln Ala Glu Gln Arg Ile Trp Asn ArgIle Glu Tyr 260 265 270 Asn Ala Ala Gly Gln Val Asp Leu Ala Glu Ala GlyAsn Gly Ile Val 275 280 285 Thr Glu Tyr Thr Tyr Glu Glu Ser Thr Gln ArgLeu Ile Arg Lys Lys 290 295 300 Asp Ser Arg Gly Leu Ser Ser Gly Glu ArgGlu Val Leu Gln Asp Tyr 305 310 315 320 Arg Tyr Glu Tyr Asp Pro Val GlyAsn Ile Leu Ser Ile Tyr Asn Glu 325 330 335 Ala Glu Pro Val Arg Tyr PheArg Asn Gln Ala Val Ala Pro Lys Arg 340 345 350 Gln Tyr Ala Tyr Asp AlaLeu Tyr Gln Leu Val Ser Ser Ser Gly Arg 355 360 365 Glu Ser Asp Ala LeuArg Gln Gln Thr Ser Leu Pro Pro Leu Ile Thr 370 375 380 Pro Ile Pro LeuAsp Asp Ser Gln Tyr Val Asn Tyr Ala Glu Lys Tyr 385 390 395 400 Ser TyrAsp Gln Ala Gly Asn Leu Ile Lys Leu Ser His Asn Gly Ala 405 410 415 SerGln Tyr Thr Thr Asn Val Tyr Val Asp Lys Ser Ser Asn Arg Gly 420 425 430Ile Trp Arg Gln Gly Glu Asp Ile Pro Asp Ile Ala Ala Ser Phe Asp 435 440445 Arg Ala Gly Asn Gln Gln Ala Leu Phe Pro Gly Arg Pro Leu Glu Trp 450455 460 Asp Thr Arg Asn Gln Leu Ser Arg Val His Met Val Val Arg Glu Gly465 470 475 480 Gly Asp Asn Asp Trp Glu Gly Tyr Leu Tyr Asp Ser Ser GlyMet Arg 485 490 495 Ile Val Lys Arg Ser Thr Arg Lys Thr Gln Thr Thr ThrGln Thr Asp 500 505 510 Thr Thr Leu Tyr Leu Pro Gly Leu Glu Leu Arg IleArg Gln Thr Gly 515 520 525 Asp Arg Val Thr Glu Ala Leu Gln Val Ile ThrVal Asp Glu Gly Ala 530 535 540 Gly Gln Val Arg Val Leu His Trp Glu AspGly Thr Glu Pro Gly Gly 545 550 555 560 Ile Ala Asn Asp Gln Tyr Arg TyrSer Leu Asn Asp His Leu Thr Ser 565 570 575 Ser Leu Leu Glu Val Asp GlyGln Gly Gln Ile Ile Ser Lys Glu Glu 580 585 590 Phe Tyr Pro Tyr Gly GlyThr Ala Leu Trp Thr Ala Arg Ser Glu Val 595 600 605 Glu Ala Ser Tyr LysThr Ile Arg Tyr Ser Gly Lys Glu Arg Asp Ala 610 615 620 Thr Gly Leu TyrTyr Tyr Gly His Arg Tyr Tyr Met Pro Trp Leu Gly 625 630 635 640 Arg TrpLeu Asn Pro Asp Pro Ala Gly Met Val Asp Gly Leu Asn Leu 645 650 655 TyrArg Met Val Arg Asn Asn Pro Ile Gly Leu Met Asp Pro Asn Gly 660 665 670Asn Ala Pro Ile Asn Val Ala Asp Tyr Ser Phe Val His Gly Asp Leu 675 680685 Val Tyr Gly Leu Ser Lys Glu Arg Gly Arg Tyr Leu Lys Leu Phe Asn 690695 700 Pro Asn Phe Asn Met Glu Lys Ser Asp Ser Pro Ala Met Val Ile Asp705 710 715 720 Gln Tyr Asn Asn Asn Val Ala Leu Ser Ile Thr Asn Gln TyrLys Val 725 730 735 Glu Glu Leu Met Lys Phe Gln Lys Asp Pro Gln Lys AlaAla Arg Lys 740 745 750 Ile Lys Val Pro Glu Gly Asn Arg Leu Ser Arg AsnGlu Asn Tyr Pro 755 760 765 Leu Trp His Asp Tyr Ile Asn Ile Gly Glu AlaLys Ala Ala Phe Lys 770 775 780 Ala Ser His Ile Phe Gln Glu Val Lys GlyAsn Tyr Gly Lys Asp Tyr 785 790 795 800 Tyr His Lys Leu Leu Leu Asp ArgMet Ile Glu Ser Pro Leu Leu Trp 805 810 815 Lys Arg Gly Ser Lys Leu GlyLeu Glu Ile Ala Ala Thr Asn Gln Arg 820 825 830 Thr Lys Ile His Phe ValLeu Asp Asn Leu Asn Ile Glu Gln Val Val 835 840 845 Thr Lys Glu Gly SerGly Gly Gln Ser Ile Thr Ala Ser Glu Leu Arg 850 855 860 Tyr Ile Tyr ArgAsn Arg Glu Arg Leu Asn Gly Arg Val Ile Phe Tyr 865 870 875 880 Arg AsnAsn Glu Arg Leu Asp Gln Ala Pro Trp Gln Glu Asn Pro Asp 885 890 895 LeuTrp Ser Lys Tyr Gln Pro Gly Leu Arg Gln Ser Ser Ser Ser Arg 900 905 910Val Lys Glu Arg Gly Ile Gly Asn Phe Phe Arg Arg Phe Ser Met Lys 915 920925 Arg Lys 930 14 1791 DNA Artificial Sequence Nucleic acid sequence ofORF7, which encodes a cry-like protein. 14 atgaactcaa atgaaccaaatttatctgat gttgttaatt gtttaagtga ccccaatagt 60 gacttggaga agtctggcggtggagtagcg ctagatgttg gaatgtcatt gatatccgaa 120 cttcttggta cggttccagttgctggatca attcttcaat ttgtattcga taaattgtgg 180 tttatttttg gcccttctgagtgggactca cttatggaac atgttgaagc attaattgat 240 agtaaaatac aagagcaggtaaaaagaagt gcacaagatg aactaaatgc aattacaaat 300 aacttatcta cgtatttgaaatttctagat gcatgggaaa atgattctaa taatttaaga 360 gcgagagctg tagtgaaagaccaatttgta ggccttgaac agactcttga aagaaaaatg 420 gttagtgttt ttggaagtacgggtcatgaa gtgcatcttt tgccaatttt cgctcaagca 480 gccaacctcc acctaattctattaagagat gctgagaaat atggaaagag atggggttgg 540 gcagatagag aaattcaagtatattatgat aaccagattc gttatatcca tgaatatacg 600 gaccattgta ttaaatattataatcaagga ttaagtaaac tgaaaggttc tacctatcaa 660 gattgggata agtataatcgttttagaaga gaaatgaccc taactgttct tgatttgatt 720 tcaattttcc catcgtatgatactagaact tacccaattg atacaatagg tcaattgaca 780 agggaagttt attcggatttacttattgct aacccgtctg ggatgcagac tttcactaat 840 gtagatttcg acaatattcttattagaaaa cctcatttaa tggatttctt aagaactctt 900 gagattttta ccgatcgacataacgcaagc agacacaacg tatattgggg cggacatcga 960 gtgcattctt cttacacaggaggtaatttt gaaaattttg aatctccctt atatggcagt 1020 gaagcaaatg tagaaccccgaacatggttg agttttggag aatctcaagt ctataatata 1080 cgttcgaagc ctgagtgggatagaggaagt actgcaatta gtggctccta tgaatttcga 1140 ggagtgacag gatgttctttttatcgaatg ggaaattttg ctggcaccgt agccctaact 1200 taccgacagt ttggtaacgaaggttctcaa atcccattgc acaggctatg tcatgttact 1260 tattttagaa gatctcaagctgtgggggcg acttcgagac agacgttaac aagtggtccg 1320 ctattttcct ggacacatagtagtgctacg gaaacgaata tcattcaccc gacaaaaatt 1380 acacaaatac caatggtgaaggctagttcc cttggatcag gtacttctgt tgtccaagga 1440 ccaggcttta caggaggggatgtacttcga agaaatagcc ccggtagcac aggaacttta 1500 agagttaacg tcaattcaccattatcacag agatatcgta taagaattcg ttacgcttct 1560 actacggatt tagatttttttgtcattcgc ggaaatacga cagttaataa ttttagattt 1620 gggaacacta tgcgtaaaggagaccctata acctctcgat catttagatt tgcggctttt 1680 agtacaccat ttacttttgctagctcacag gatgaactta gaataaatgt acaaaatttc 1740 aataatggtg aagaagtttatatagataga atcgaagtta ttccagtttg a 1791 15 596 PRT Artificial SequenceAmino acid sequence encoded by ORF7. 15 Met Asn Ser Asn Glu Pro Asn LeuSer Asp Val Val Asn Cys Leu Ser 1 5 10 15 Asp Pro Asn Ser Asp Leu GluLys Ser Gly Gly Gly Val Ala Leu Asp 20 25 30 Val Gly Met Ser Leu Ile SerGlu Leu Leu Gly Thr Val Pro Val Ala 35 40 45 Gly Ser Ile Leu Gln Phe ValPhe Asp Lys Leu Trp Phe Ile Phe Gly 50 55 60 Pro Ser Glu Trp Asp Ser LeuMet Glu His Val Glu Ala Leu Ile Asp 65 70 75 80 Ser Lys Ile Gln Glu GlnVal Lys Arg Ser Ala Gln Asp Glu Leu Asn 85 90 95 Ala Ile Thr Asn Asn LeuSer Thr Tyr Leu Lys Phe Leu Asp Ala Trp 100 105 110 Glu Asn Asp Ser AsnAsn Leu Arg Ala Arg Ala Val Val Lys Asp Gln 115 120 125 Phe Val Gly LeuGlu Gln Thr Leu Glu Arg Lys Met Val Ser Val Phe 130 135 140 Gly Ser ThrGly His Glu Val His Leu Leu Pro Ile Phe Ala Gln Ala 145 150 155 160 AlaAsn Leu His Leu Ile Leu Leu Arg Asp Ala Glu Lys Tyr Gly Lys 165 170 175Arg Trp Gly Trp Ala Asp Arg Glu Ile Gln Val Tyr Tyr Asp Asn Gln 180 185190 Ile Arg Tyr Ile His Glu Tyr Thr Asp His Cys Ile Lys Tyr Tyr Asn 195200 205 Gln Gly Leu Ser Lys Leu Lys Gly Ser Thr Tyr Gln Asp Trp Asp Lys210 215 220 Tyr Asn Arg Phe Arg Arg Glu Met Thr Leu Thr Val Leu Asp LeuIle 225 230 235 240 Ser Ile Phe Pro Ser Tyr Asp Thr Arg Thr Tyr Pro IleAsp Thr Ile 245 250 255 Gly Gln Leu Thr Arg Glu Val Tyr Ser Asp Leu LeuIle Ala Asn Pro 260 265 270 Ser Gly Met Gln Thr Phe Thr Asn Val Asp PheAsp Asn Ile Leu Ile 275 280 285 Arg Lys Pro His Leu Met Asp Phe Leu ArgThr Leu Glu Ile Phe Thr 290 295 300 Asp Arg His Asn Ala Ser Arg His AsnVal Tyr Trp Gly Gly His Arg 305 310 315 320 Val His Ser Ser Tyr Thr GlyGly Asn Phe Glu Asn Phe Glu Ser Pro 325 330 335 Leu Tyr Gly Ser Glu AlaAsn Val Glu Pro Arg Thr Trp Leu Ser Phe 340 345 350 Gly Glu Ser Gln ValTyr Asn Ile Arg Ser Lys Pro Glu Trp Asp Arg 355 360 365 Gly Ser Thr AlaIle Ser Gly Ser Tyr Glu Phe Arg Gly Val Thr Gly 370 375 380 Cys Ser PheTyr Arg Met Gly Asn Phe Ala Gly Thr Val Ala Leu Thr 385 390 395 400 TyrArg Gln Phe Gly Asn Glu Gly Ser Gln Ile Pro Leu His Arg Leu 405 410 415Cys His Val Thr Tyr Phe Arg Arg Ser Gln Ala Val Gly Ala Thr Ser 420 425430 Arg Gln Thr Leu Thr Ser Gly Pro Leu Phe Ser Trp Thr His Ser Ser 435440 445 Ala Thr Glu Thr Asn Ile Ile His Pro Thr Lys Ile Thr Gln Ile Pro450 455 460 Met Val Lys Ala Ser Ser Leu Gly Ser Gly Thr Ser Val Val GlnGly 465 470 475 480 Pro Gly Phe Thr Gly Gly Asp Val Leu Arg Arg Asn SerPro Gly Ser 485 490 495 Thr Gly Thr Leu Arg Val Asn Val Asn Ser Pro LeuSer Gln Arg Tyr 500 505 510 Arg Ile Arg Ile Arg Tyr Ala Ser Thr Thr AspLeu Asp Phe Phe Val 515 520 525 Ile Arg Gly Asn Thr Thr Val Asn Asn PheArg Phe Gly Asn Thr Met 530 535 540 Arg Lys Gly Asp Pro Ile Thr Ser ArgSer Phe Arg Phe Ala Ala Phe 545 550 555 560 Ser Thr Pro Phe Thr Phe AlaSer Ser Gln Asp Glu Leu Arg Ile Asn 565 570 575 Val Gln Asn Phe Asn AsnGly Glu Glu Val Tyr Ile Asp Arg Ile Glu 580 585 590 Val Ile Pro Val 59516 1547 DNA Artificial Sequence Nucleic acid sequence of the 16S rDNA ofIDAS1529. 16 tggagagttt gatcctggct caggacgaac gctggcggcg tgcctaatacatgcaagtcg 60 agcggakcaa cggtttcctt cgggaaaccr ttagcttagc ggcggacgggtgagtaatac 120 gtaggtaacc tgcccttaag accgggataa ctcacggaaa cgtgggctaataccggatag 180 gcgatttcct cgcatgaggg aatcgggaaa ggcggagcaa tctgccacttatggatggac 240 ctacggcgca ttagctagtt ggtggggtaa cggctcacca aggcgacgatgcgtagccga 300 cctgagaggg tgatcggcca cactgggact gagacacggc ccagactcctacgggaggca 360 gcagtaggga atcttccgca atggacgcaa gtctgacgga gcaacgccgcgtgagtgatg 420 aaggttttcg gatcgtaaag ctctgttgcc agggaagaac gctatggagagtaactgttc 480 cataggtgac ggtacctgag aagaaagccc cggctaacta cgtgccagcagccgcggtaa 540 tacgtagggg gcaagcgttg tccggaatta ttgggcgtaa agcgcgcgcaggcggtcatg 600 taagtctggt gtttaaaccc ggggctcaac tccgggtcgc atcggaaactgtgtgacttg 660 agtgcagaag aggaaagtgg aattccacgt gtagcggtga aatgcgtagagatgtggagg 720 aacaccagtg gcgaaggcga ctttctgggc tgtaactgac gctgaggcgcgaaagcgtgg 780 ggagcaaaca ggattagata ccctggtagt ccacgccgta aacgatgaatgctaggtgtt 840 aggggtttcg atacccttgg tgccgaagtt aacacattaa gcattccgcctggggagtac 900 ggtcgcaaga ctgaaactca aaggaattga cggggacccg cacaagcagtggagtatgtg 960 gtttaattcg aagcaacgcg aagaacctta ccaggtcttg acatccctctgaccgtccta 1020 gagatagggc ttcccttcgg ggcagaggag acaggtggtg catggttgtcgtcagctcgt 1080 gtcgtgagat gttgggttaa gtcccgcaac gagcgcaacc cttaactttagttgccagca 1140 ttaagttggg cactctagag tgactgccgg tgacaaaccg gaggaaggtggggatgacgt 1200 caaatcatca tgccccttat gacctgggct acacacgtac tacaatggctggtacaacgg 1260 gaagcgaagc cgcgaggtgg agcgaatcct aaaaagccag tctcagttcggattgcaggc 1320 tgcaactcgc ctgcatgaag tcggaattgc tagtaatcgc ggatcagcatgccgcggtga 1380 atacgttccc gggtcttgta cacaccgccc gtcacaccac gagagtttacaacacccgaa 1440 gtcggtgggg taaccgcaag gagccagccg ccgaaggtgg ggtagatgattggggtgaag 1500 tcgtaacaag gtagccgtat cggaaggtgc ggytggatca cctcctt 154717 20 PRT Artificial Sequence N-terminal amino acid sequence for thepurified toxin from the broth fraction from IDAS1529. 17 Asp Ile Thr LeuLys Val Ala Ile Tyr Pro Tyr Val Pro Asp Pro Ser 1 5 10 15 Arg Phe GlnAla 20 18 379 PRT Artificial Sequence Amino acid sequence of thiaminaseI from Bacillus thiaminolyticus. 18 Ala His Ser Asp Ala Ser Ser Asp IleThr Leu Lys Val Ala Ile Tyr 1 5 10 15 Pro Tyr Val Pro Asp Pro Ala ArgPhe Gln Ala Ala Val Leu Asp Gln 20 25 30 Trp Gln Arg Gln Glu Pro Gly ValLys Leu Glu Phe Thr Asp Trp Asp 35 40 45 Ser Tyr Ser Ala Asp Pro Pro AspAsp Leu Asp Val Phe Val Leu Asp 50 55 60 Ser Ile Phe Leu Ser His Phe ValAsp Ala Gly Tyr Leu Leu Pro Phe 65 70 75 80 Gly Ser Gln Asp Ile Asp GlnAla Glu Asp Val Leu Pro Phe Ala Leu 85 90 95 Gln Gly Ala Lys Arg Asn GlyGlu Val Tyr Gly Leu Pro Gln Ile Leu 100 105 110 Cys Thr Asn Leu Leu PheTyr Arg Lys Gly Asp Leu Lys Ile Gly Gln 115 120 125 Val Asp Asn Ile TyrGlu Leu Tyr Lys Lys Ile Gly Thr Ser His Ser 130 135 140 Glu Gln Ile ProPro Pro Gln Asn Lys Gly Leu Leu Ile Asn Met Ala 145 150 155 160 Gly GlyThr Thr Lys Ala Ser Met Tyr Leu Glu Ala Leu Ile Asp Val 165 170 175 ThrGly Gln Tyr Thr Glu Tyr Asp Leu Leu Pro Pro Leu Asp Pro Leu 180 185 190Asn Asp Lys Val Ile Arg Gly Leu Arg Leu Leu Ile Asn Met Ala Gly 195 200205 Glu Lys Pro Ser Gln Tyr Val Pro Glu Asp Gly Asp Ala Tyr Val Arg 210215 220 Ala Ser Trp Phe Ala Gln Gly Ser Gly Arg Ala Phe Ile Gly Tyr Ser225 230 235 240 Glu Ser Met Met Arg Met Gly Asp Tyr Ala Glu Gln Val ArgPhe Lys 245 250 255 Pro Ile Ser Ser Ser Ala Gly Gln Asp Ile Pro Leu PheTyr Ser Asp 260 265 270 Val Val Ser Val Asn Ser Lys Thr Ala His Pro GluLeu Ala Lys Lys 275 280 285 Leu Ala Asn Val Met Ala Ser Ala Asp Thr ValGlu Gln Ala Leu Arg 290 295 300 Pro Gln Ala Asp Gly Gln Tyr Pro Gln TyrLeu Leu Pro Ala Arg His 305 310 315 320 Gln Val Tyr Glu Ala Leu Met GlnAsp Tyr Pro Ile Tyr Ser Glu Leu 325 330 335 Ala Gln Ile Val Asn Lys ProSer Asn Arg Val Phe Arg Leu Gly Pro 340 345 350 Glu Val Arg Thr Trp LeuLys Asp Ala Lys Gln Val Leu Pro Glu Ala 355 360 365 Leu Gly Leu Thr AspVal Ser Ser Leu Ala Ser 370 375 19 953 PRT Paenibacillus strain IDAS1529 19 Met Lys Met Ile Pro Trp Thr His His Tyr Leu Leu His Arg Leu Arg1 5 10 15 Gly Glu Met Glu Val Lys Pro Met Asn Thr Thr Ser Ile Tyr ArgGly 20 25 30 Thr Pro Thr Ile Ser Val Val Asp Asn Arg Asn Leu Glu Ile ArgIle 35 40 45 Leu Gln Tyr Asn Arg Ile Ala Ala Glu Asp Pro Ala Asp Glu CysIle 50 55 60 Leu Arg Asn Thr Tyr Thr Pro Leu Ser Tyr Leu Gly Ser Ser MetAsp 65 70 75 80 Pro Arg Leu Phe Ser Gln Tyr Gln Asp Asp Arg Gly Thr ProPro Asn 85 90 95 Ile Arg Thr Met Ala Ser Leu Arg Gly Glu Ala Leu Cys SerGlu Ser 100 105 110 Val Asp Ala Gly Arg Lys Ala Glu Leu Phe Asp Ile GluGly Arg Pro 115 120 125 Val Trp Leu Ile Asp Ala Asn Gly Thr Glu Thr ThrLeu Glu Tyr Asp 130 135 140 Val Leu Gly Arg Pro Thr Ala Val Phe Glu GlnGln Glu Gly Thr Asp 145 150 155 160 Ser Pro Gln Cys Arg Glu Arg Phe IleTyr Gly Glu Lys Glu Ala Asp 165 170 175 Ala Gln Ala Asn Asn Leu Arg GlyGln Leu Val Arg His Tyr Asp Thr 180 185 190 Ala Gly Arg Ile Gln Thr AspSer Ile Ser Leu Ala Gly Leu Pro Leu 195 200 205 Arg Gln Ser Arg Gln LeuLeu Lys Asn Trp Asp Glu Pro Gly Asp Trp 210 215 220 Ser Met Asp Glu GluSer Ala Trp Ala Ser Leu Leu Ala Ala Glu Ala 225 230 235 240 Tyr Asp ThrSer Trp Arg Tyr Asp Ala Gln Asp Arg Val Leu Ala Gln 245 250 255 Thr AspAla Lys Gly Asn Leu Gln Gln Leu Thr Tyr Asn Asp Ala Gly 260 265 270 GlnPro Gln Ala Val Ser Leu Lys Leu Gln Gly Gln Ala Glu Gln Arg 275 280 285Ile Trp Asn Arg Ile Glu Tyr Asn Ala Ala Gly Gln Val Asp Leu Ala 290 295300 Glu Ala Gly Asn Gly Ile Val Thr Glu Tyr Thr Tyr Glu Glu Ser Thr 305310 315 320 Gln Arg Leu Ile Arg Lys Lys Asp Ser Arg Gly Leu Ser Ser GlyGlu 325 330 335 Arg Glu Val Leu Gln Asp Tyr Arg Tyr Glu Tyr Asp Pro ValGly Asn 340 345 350 Ile Leu Ser Ile Tyr Asn Glu Ala Glu Pro Val Arg TyrPhe Arg Asn 355 360 365 Gln Ala Val Ala Pro Lys Arg Gln Tyr Ala Tyr AspAla Leu Tyr Gln 370 375 380 Leu Val Ser Ser Ser Gly Arg Glu Ser Asp AlaLeu Arg Gln Gln Thr 385 390 395 400 Ser Leu Pro Pro Leu Ile Thr Pro IlePro Leu Asp Asp Ser Gln Tyr 405 410 415 Val Asn Tyr Ala Glu Lys Tyr SerTyr Asp Gln Ala Gly Asn Leu Ile 420 425 430 Lys Leu Ser His Asn Gly AlaSer Gln Tyr Thr Thr Asn Val Tyr Val 435 440 445 Asp Lys Ser Ser Asn ArgGly Ile Trp Arg Gln Gly Glu Asp Ile Pro 450 455 460 Asp Ile Ala Ala SerPhe Asp Arg Ala Gly Asn Gln Gln Ala Leu Phe 465 470 475 480 Pro Gly ArgPro Leu Glu Trp Asp Thr Arg Asn Gln Leu Ser Arg Val 485 490 495 His MetVal Val Arg Glu Gly Gly Asp Asn Asp Trp Glu Gly Tyr Leu 500 505 510 TyrAsp Ser Ser Gly Met Arg Ile Val Lys Arg Ser Thr Arg Lys Thr 515 520 525Gln Thr Thr Thr Gln Thr Asp Thr Thr Leu Tyr Leu Pro Gly Leu Glu 530 535540 Leu Arg Ile Arg Gln Thr Gly Asp Arg Val Thr Glu Ala Leu Gln Val 545550 555 560 Ile Thr Val Asp Glu Gly Ala Gly Gln Val Arg Val Leu His TrpGlu 565 570 575 Asp Gly Thr Glu Pro Gly Gly Ile Ala Asn Asp Gln Tyr ArgTyr Ser 580 585 590 Leu Asn Asp His Leu Thr Ser Ser Leu Leu Glu Val AspGly Gln Gly 595 600 605 Gln Ile Ile Ser Lys Glu Glu Phe Tyr Pro Tyr GlyGly Thr Ala Leu 610 615 620 Trp Thr Ala Arg Ser Glu Val Glu Ala Ser TyrLys Thr Ile Arg Tyr 625 630 635 640 Ser Gly Lys Glu Arg Asp Ala Thr GlyLeu Tyr Tyr Tyr Gly His Arg 645 650 655 Tyr Tyr Met Pro Trp Leu Gly ArgTrp Leu Asn Pro Asp Pro Ala Gly 660 665 670 Met Val Asp Gly Leu Asn LeuTyr Arg Met Val Arg Asn Asn Pro Ile 675 680 685 Gly Leu Met Asp Pro AsnGly Asn Ala Pro Ile Asn Val Ala Asp Tyr 690 695 700 Ser Phe Val His GlyAsp Leu Val Tyr Gly Leu Ser Lys Glu Arg Gly 705 710 715 720 Arg Tyr LeuLys Leu Phe Asn Pro Asn Phe Asn Met Glu Lys Ser Asp 725 730 735 Ser ProAla Met Val Ile Asp Gln Tyr Asn Asn Asn Val Ala Leu Ser 740 745 750 IleThr Asn Gln Tyr Lys Val Glu Glu Leu Met Lys Phe Gln Lys Asp 755 760 765Pro Gln Lys Ala Ala Arg Lys Ile Lys Val Pro Glu Gly Asn Arg Leu 770 775780 Ser Arg Asn Glu Asn Tyr Pro Leu Trp His Asp Tyr Ile Asn Ile Gly 785790 795 800 Glu Ala Lys Ala Ala Phe Lys Ala Ser His Ile Phe Gln Glu ValLys 805 810 815 Gly Asn Tyr Gly Lys Asp Tyr Tyr His Lys Leu Leu Leu AspArg Met 820 825 830 Ile Glu Ser Pro Leu Leu Trp Lys Arg Gly Ser Lys LeuGly Leu Glu 835 840 845 Ile Ala Ala Thr Asn Gln Arg Thr Lys Ile His PheVal Leu Asp Asn 850 855 860 Leu Asn Ile Glu Gln Val Val Thr Lys Glu GlySer Gly Gly Gln Ser 865 870 875 880 Ile Thr Ala Ser Glu Leu Arg Tyr IleTyr Arg Asn Arg Glu Arg Leu 885 890 895 Asn Gly Arg Val Ile Phe Tyr ArgAsn Asn Glu Arg Leu Asp Gln Ala 900 905 910 Pro Trp Gln Glu Asn Pro AspLeu Trp Ser Lys Tyr Gln Pro Gly Leu 915 920 925 Arg Gln Ser Ser Ser SerArg Val Lys Glu Arg Gly Ile Gly Asn Phe 930 935 940 Phe Arg Arg Phe SerMet Lys Arg Lys 945 950 20 4482 DNA Xenorhabdus strain Xwi 20 atgcagggttcaacaccttt gaaacttgaa ataccgtcat tgccctctgg gggcggatca 60 ctaaaaggaatgggagaagc actcaatgcc gtcggagcgg aagggggagc gtcattttca 120 ctgcccttgccgatctctgt cgggcgtggt ctggtgccgg tgctatcact gaattacagc 180 agtactgccggcaatgggtc attcgggatg gggtggcaat gtggggttgg ttttatcagc 240 ctgcgtaccgccaagggcgt tccgcactat acgggacaag atgagtatct cgggccggat 300 ggggaagtgttgagtattgt gccggacagc caagggcaac cagagcaacg caccgcaacc 360 tcactgttggggacggttct gacacagccg catactgtta cccgctatca gtcccgcgtg 420 gcagaaaaaatcgttcgttt agaacactgg cagccacagc agagacgtga ggaagagacg 480 tctttttgggtactttttac tgcggatggt ttagtgcacc tattcggtaa gcatcaccat 540 gcacgtattgctgacccgca ggatgaaacc agaattgccc gctggctgat ggaggaaacc 600 gtcacgcataccggggaaca tatttactat cactatcggg cagaagacga tcttgactgt 660 gatgagcatgaacttgctca gcattcaggt gttacggccc agcgttatct ggcaaaagtc 720 agctatggcaatactcagcc ggaaaccgct tttttcgcgg taaaatcagg tattcctgct 780 gataatgactggctgtttca tctggtattt gattacggtg agcgctcatc ttcgctgaac 840 tctgtacccgaattcaatgt gtcagaaaac aatgtgtctg aaaacaatgt gcctgaaaaa 900 tggcgttgtcgtccggacag tttctcccgc tatgaatatg ggtttgaaat tcgaacccgt 960 cgcttgtgtcgccaagttct gatgtttcat cagctgaaag cgctggcagg ggaaaaggtt 1020 gcagaagaaacaccggcgct ggtttcccgt cttattctgg attatgacct gaacaacaag 1080 gtttccttgctgcaaacggc ccgcagactg gcccatgaaa cggacggtac gccagtgatg 1140 atgtccccgctggaaatgga ttatcaacgt gttaatcatg gcgtgaatct gaactggcag 1200 tccatgccgcagttagaaaa aatgaacacg ttgcagccat accaattggt tgatttatat 1260 ggagaaggaatttccggcgt actttatcag gatactcaga aagcctggtg gtaccgtgct 1320 ccggtacgggatatcactgc cgaaggaacg aatgcggtta cctatgagga ggccaaacca 1380 ctgccacatattccggcaca acaggaaagc gcgatgttgt tggacatcaa tggtgacggg 1440 cgtctggattgggtgattac ggcatcaggg ttacggggct accacaccat gtcaccggaa 1500 ggtgaatggacaccctttat tccattatcc gctgtgccaa tggaatattt ccatccgcag 1560 gcaaaactggctgatattga tggggctggg ctgcctgact tagcgcttat cgggccaaat 1620 agtgtacgtgtctggtcaaa taatcgggca ggatgggatc gcgctcagga tgtgattcat 1680 ttgtcagatatgccactgcc ggttcccggc agaaatgagc gtcatcttgt cgcattcagt 1740 gatatgacaggctccgggca atcacatctg gtggaagtaa cggcagatag cgtgcgctac 1800 tggccgaacctggggcatgg aaaatttggt gagcctctga tgatgacagg cttccagatt 1860 agcggggaaacgtttaaccc cgacagactg tatatggtag acatagatgg ctcaggcacc 1920 accgattttatttatgcccg caatacttac cttgaactct atgccaatga aagcggcaat 1980 cattttgctgaacctcagcg tattgatctg ccggatgggg tacgttttga tgatacttgt 2040 cggttacaaatagcggatac acaaggatta gggactgcca gcattatttt gacgatcccc 2100 catatgaaggtgcagcactg gcgattggat atgaccatat tcaagccttg gctgctgaat 2160 gccgtcaataacaatatggg aacagaaacc acgctgtatt atcgcagctc tgcccagttc 2220 tggctggatgagaaattaca ggcttctgaa tccgggatga cggtggtcag ctacttaccg 2280 ttcccggtgcatgtgttgtg gcgcacggaa gtgctggatg aaatttccgg taaccgattg 2340 accagccattatcattactc acatggtgcc tgggatggtc tggaacggga gtttcgtggt 2400 tttgggcgggtgacacaaac tgatattgat tcacgggcga gtgcgacaca ggggacacat 2460 gctgaaccaccggcaccttc gcgcacggtt aattggtacg gcactggcgt acgggaagtc 2520 gatattcttctgcccacgga atattggcag ggggatcaac aggcatttcc ccattttacc 2580 ccacgctttacccgttatga cgaaaaatcc ggtggtgata tgacggtcac gccgagcgaa 2640 caggaagaatactggttaca tcgagcctta aaaggacaac gtttacgcag tgagctgtat 2700 ggggatgatgattctatact ggccggtacg ccttattcag tggatgaatc ccgcacccaa 2760 gtacgtttgttaccggtgat ggtatcggac gtgcctgcgg tactggtttc ggtggccgaa 2820 tcccgccaataccgatatga acgggttgct accgatccac agtgcagcca aaagatcgtc 2880 cttaaatctgatgcgttagg atttccgcag gacaatcttg agattgccta ttcgagacgt 2940 ccacagcctgagttctcgcc ttatccggat accctgcccg aaacactttt caccagcagt 3000 ttcgacgaacagcagatgtt ccttcgtctg acacgccagc gttcttctta tcatcatctg 3060 aatcatgatgataatacgtg gatcacaggg cttatggata cctcacgcag tgacgcacgt 3120 atttatcaagccgataaagt gccggacggt ggattttccc ttgaatggtt ttctgccaca 3180 ggtgcaggagcattgttgtt gcctgatgcc gcagccgatt atctgggaca tcagcgtgta 3240 gcatataccggtccagaaga acaacccgct attcctccgc tggtggcata cattgaaacc 3300 gcagagtttgatgaacgatc gttggcggct tttgaggagg tgatggatga gcaggagctg 3360 acaaaacagctgaatgatgc gggctggaat acggcaaaag tgccgttcag tgaaaagaca 3420 gatttccatgtctgggtggg acaaaaggaa tttacagaat atgccggtgc agacggattc 3480 tatcggccattggtgcaacg ggaaaccaag cttacaggta aaacgacagt cacgtgggat 3540 agccattactgtgttatcac cgcaacagag gatgcggctg gcctgcgtat gcaagcgcat 3600 tacgattatcgatttatggt tgcggataac accacagatg tcaatgataa ctatcacacc 3660 gtgacgtttgatgcactggg gagggtaacc agcttccgtt tctgggggac tgaaaacggt 3720 gaaaaacaaggatatacccc tgcggaaaat gaaactgtcc cctttattgt ccccacaacg 3780 gtggatgatgctctggcatt gaaacccggt atacctgttg cagggctgat ggtttatgcc 3840 cctctgagctggatggttca ggccagcttt tctaatgatg gggagcttta tggagagctg 3900 aaaccggctgggatcatcac tgaagatggt tatctcctgt cgcttgcttt tcgccgctgg 3960 caacaaaataaccctgccgc tgccatgcca aagcaagtca attcacagaa cccaccccat 4020 gtactgagtgtgatcaccga ccgctatgat gccgatccgg aacaacaatt acgtcaaacg 4080 tttacgtttagtgatggttt tgggcgaacc ttacaaacag ccgtacgcca tgaaagtggt 4140 gaagcctgggtacgtgatga gtatggagcc attgtggctg aaaatcatgg cgcgcctgaa 4200 acggcgatgacagatttccg ttgggcagtt tccggacgta cagaatatga cggaaaaggc 4260 caagccctgcgtaagtatca accgtatttc ctgaatagtt ggcagtacgt cagtgatgac 4320 agtgcccggcaggatatata tgccgatacc cattactatg atccgttggg gcgtgaatat 4380 caggttatcacggccaaagg cgggtttcgt cgatccttat tcactccctg gtttgtggtg 4440 aatgaagatgaaaatgacac tgccggtgaa atgacagcat aa 4482 21 3051 DNA Xenorhabdus strainXwi 21 atgaagaatt tcgttcacag caatacgcca tccgtcaccg tactggacaa ccgtggtcag60 acagtacgcg aaatagcctg gtatcggcac cccgatacac ctcaggtaac cgatgaacgc 120atcaccggtt atcaatatga tgctcaagga tctctgactc agagtattga tccgcgattt 180tatgaacgcc agcagacagc gagtgacaag aacgccatta cacccaatct tattctcttg 240tcatcactca gtaagaaggc attgcgtacg caaagtgtgg atgccggaac ccgtgtcgcc 300ctgcatgatg ttgccgggcg tcccgtttta gctgtcagcg ccaatggcgt tagccgaacg 360tttcagtatg aaagtgataa ccttccggga cgattgctaa cgattaccga gcaggtaaaa 420ggagagaacg cctgtatcac ggagcgattg atctggtcag gaaatacgcc ggcagaaaaa 480ggcaataatc tggccggcca gtgcgtggtc cattatgatc ccaccggaat gaatcaaacc 540aacagcatat cgttaaccag catacccttg tccatcacac agcaattact gaaagatgac 600agcgaagccg attggcacgg tatggatgaa tctggctgga aaaacgcgct ggcgccggaa 660agcttcactt ctgtcagcac aacggatgct accggcacgg tattaacgag tacagatgct 720gccggaaaca agcaacgtat cgcctatgat gtggccggtc tgcttcaagg cagttggttg 780gcgctgaagg ggaaacaaga acaagttatc gtgaaatccc tgacctattc ggctgccagc 840cagaagctac gggaggaaca tggtaacggg atagtgacta catataccta tgaacccgag 900acgcaacgag ttattggcat aaaaacagaa cgtccttccg gtcatgccgc tggggagaaa 960attttacaaa acctgcgtta tgaatatgat cctgtcggaa atgtgctgaa atcaactaat 1020gatgctgaaa ttacccgctt ttggcgcaac cagaaaattg taccggaaaa tacttacacc 1080tatgacagcc tgtaccagct ggtttccgtc actgggcgtg aaatggcgaa tattggccga 1140caaaaaaacc agttacccat ccccgctctg attgataaca atacttatac gaattactct 1200cgcacttacg actatgatcg tgggggaaat ctgaccagaa ttcgccataa ttcaccgatc 1260accggtaata actatacaac gaacatgacc gtttcagatc acagcaaccg ggctgtactg 1320gaagagctgg cgcaagatcc cactcaggtg gatatgttgt tcacccccgg cgggcatcag 1380acccggcttg ttcccggtca ggatcttttc tggacacccc gtgacgaatt gcaacaagtg 1440atattggtca atagggaaaa tacgacgcct gatcaggaat tctaccgtta tgatgcagac 1500agtcagcgtg tcattaagac tcatattcag aagacaggta acagtgagca aatacagcga 1560acattatatt tgccagagct ggaatggcgc acgacatata gcggcaatac attaaaagag 1620tttttgcagg tcatcactgt cggtgaatcg ggtcaggcac aagtgcgggt gctgcattgg 1680gaaacaggca aaccggcgga tatcagcaat gatcagctgc gctacagtta tggcaacctg 1740attggcagta gcgggctgga attggacagt gacgggcaga tcattagtca ggaagaatat 1800tacccctatg ggggaaccgc cgtgtgggca gcccgaagtc agtcagaagc tgattacaaa 1860accgtgcgtt attctggcaa agagcgggat gcaacagggt tgtattacta cggttatcgt 1920tattatcaat cgtggacagg gcgatggttg agtgtagatc ctgccggtga ggtcgatggt 1980ctcaatttgt tccgaatgtg caggaataac cccatcgttt tttctgattc tgatggtcgt 2040ttccccggtc agggtgtcct tgcctggata gggaaaaaag cgtatcgaaa ggcagtcaac 2100atcacgacag aacacctgct tgaacaaggc gcttcctttg atacgttctt gaaattaaac 2160cgaggattgc gaacgtttgt tttgggtgtg ggggtagcaa gtctgggggt gaaggcggcc 2220acgattgcag gagcgtcgcc ttgggggatt gtcggggctg ccattggtgg ttttgtctcc 2280ggggcggtga tggggttttt cgcgaacaac atctcagaaa aaattgggga agttttaagt 2340tatctgacgc gtaaacgttc tgttcctgtt caggttggcg cttttgttgt cacatcgctt 2400gtgacgtctg cactatttaa cagctcttcg acaggtaccg ccatttccgc agcaacagcg 2460gtcaccgttg gaggattaat ggctttagcc ggagagcata acacgggcat ggctatcagt 2520attgccacac ccgccggaca aggtacgctg gatacgctca ggcccggtaa tgtcagcgcg 2580ccagagcggt taggggcact atcaggcgca attattggcg gcatattact tggccgccat 2640cagggaagtt ctgagctggg tgaacgggca gcgattggtg ctatgtatgg tgctcgatgg 2700ggaaggatca ttggtaatct atgggatggc ccttatcggt ttatcggcag gttactgctc 2760agaagaggca ttagctctgc catttcccac gctgtcagtt ccaggagctg gtttggccga 2820atgataggag aaagtgtcgg gagaaatatt tctgaagtat tattacctta tagccgtaca 2880cccggtgaat gggttggtgc agccattggc gggacagccg cggccgctca tcatgccgtt 2940ggaggggaag ttgccaatgc cgctagccgg gttacctgga gcggctttaa gcgggctttt 3000aataacttct tctttaacgc ctctgcacgt cataatgaat ccgaagcata a 3051 22 32 DNAArtificial Sequence Primer SB101 22 gckatggcsg acccgatgca wtacaagctg gc32 23 32 DNA Artificial Sequence Primer SB102 23 agcggytgac crtccagrctcarattgtgg cg 32 24 28 DNA Artificial Sequence Primer SB103 24tgtataactg gatggcyggw cgtctstc 28 25 26 DNA Artificial Sequence PrimerSB104 25 tcraaaggca graamcggct gtcgtt 26 26 28 DNA Artificial SequencePrimer SB105 26 cttcyctkga tatcytkytg gatgtgct 28 27 30 DNA ArtificialSequence Primer SB106 27 acgrctggya ttggyaatca gccartccaa 30 28 27 DNAArtificial Sequence Primer SB212 28 cgytatnaat atgaycckgt vggyaat 27 2925 DNA Artificial Sequence Primer SB213 29 catcbcgytc tttrccngar tarcg25 30 33 DNA Artificial Sequence Primer SB215 30 cghagctcyn cccagtwytggctggatgar aaa 33 31 32 DNA Artificial Sequence Primer SB217 31gtrtcatttt catcttcrtt bacnryaaac ca 32 32 1293 DNA Paenibacillus apairusstrain DB482 32 gcagcccgaa ggctccggca cgctggcatc cttgaaggat acctaccatccgatgaccct 60 tccctatgat gacgaccttg cgcaaatcaa tgccgtggcg gaggcgcactcatctaattt 120 gctggggatt tgggataccc tgctggacac gcagcggact tccatcctgcagaattccgc 180 cgctgcctgc cggataagca aggcgcggca atcggcatcc ccggatcagagagcctccga 240 tgatgagccg gtattgatta caggagaaga attctacctg gagacgggcggcaaacggct 300 ttttctggcg cataaactcg agataggctc cacgataagc gccaaaatcaacattggacc 360 gccgcaagcg gccgatatcg cgccagcaaa gttgcaactc gtttattacggcagaggcgg 420 cagaggggac tacttccttc gtgtggcaga cgatgtgtcc ctcggtggaaaattgctgaa 480 caattgttat ctgaccagcg acgacggaca gagcaacaat attaacggaccattctgcct 540 aatgattaat cgaggcaccg gcagcatgcc cagcgggact cacctgccagttcagattga 600 cagagtgaca gatacatccc tacgcatttt tgtgccgcaa cacggttacttgggactagg 660 agaaagcctt gccagcaact ggaatgaacc gttggcgctg aatctggacttggatcaagc 720 gttgaccttt accctaagaa agaatgagtc cggacaagat accatttccataatcgatat 780 gatgccgcct gttgccgaca cgaccccgtc cccgccgacg agggaaacgctttccttgac 840 gccaaacagc ttccgtctgc tggttaaccc cgagccgaca gaagaagacatcgccaagca 900 ctacaacgtt aagactgcca taacccgagc tcctgccgat ctggccgccgccttaaatgt 960 tgtcgatgat ttctgcatga agaccggctt gagctttgat gaattgctgaacttaacgat 1020 gcagaaggat tatcagtcaa aaagcagtga gtacaaaagc cgatttgtaaaatttggcgg 1080 cggggagcat gttccggttt caacctatgg agctgtgttt ttgacaggtacggaagaaac 1140 tccgttgtgg gcaaaacagt ataacagcgc aggcgctgca acagacacccctgttttgaa 1200 ctttacggcg gataatgttg cagctttggc aggaagagcg gaaaagcttgtgcggctggc 1260 gcgaagcacg ggtctttcct ttgagcagtt gga 1293 33 430 PRTPaenibacillus apairius strain DB482 33 Gln Pro Glu Gly Ser Gly Thr LeuAla Ser Leu Lys Asp Thr Tyr His 1 5 10 15 Pro Met Thr Leu Pro Tyr AspAsp Asp Leu Ala Gln Ile Asn Ala Val 20 25 30 Ala Glu Ala His Ser Ser AsnLeu Leu Gly Ile Trp Asp Thr Leu Leu 35 40 45 Asp Thr Gln Arg Thr Ser IleLeu Gln Asn Ser Ala Ala Ala Cys Arg 50 55 60 Ile Ser Lys Ala Arg Gln SerAla Ser Pro Asp Gln Arg Ala Ser Asp 65 70 75 80 Asp Glu Pro Val Leu IleThr Gly Glu Glu Phe Tyr Leu Glu Thr Gly 85 90 95 Gly Lys Arg Leu Phe LeuAla His Lys Leu Glu Ile Gly Ser Thr Ile 100 105 110 Ser Ala Lys Ile AsnIle Gly Pro Pro Gln Ala Ala Asp Ile Ala Pro 115 120 125 Ala Lys Leu GlnLeu Val Tyr Tyr Gly Arg Gly Gly Arg Gly Asp Tyr 130 135 140 Phe Leu ArgVal Ala Asp Asp Val Ser Leu Gly Gly Lys Leu Leu Asn 145 150 155 160 AsnCys Tyr Leu Thr Ser Asp Asp Gly Gln Ser Asn Asn Ile Asn Gly 165 170 175Pro Phe Cys Leu Met Ile Asn Arg Gly Thr Gly Ser Met Pro Ser Gly 180 185190 Thr His Leu Pro Val Gln Ile Asp Arg Val Thr Asp Thr Ser Leu Arg 195200 205 Ile Phe Val Pro Gln His Gly Tyr Leu Gly Leu Gly Glu Ser Leu Ala210 215 220 Ser Asn Trp Asn Glu Pro Leu Ala Leu Asn Leu Asp Leu Asp GlnAla 225 230 235 240 Leu Thr Phe Thr Leu Arg Lys Asn Glu Ser Gly Gln AspThr Ile Ser 245 250 255 Ile Ile Asp Met Met Pro Pro Val Ala Asp Thr ThrPro Ser Pro Pro 260 265 270 Thr Arg Glu Thr Leu Ser Leu Thr Pro Asn SerPhe Arg Leu Leu Val 275 280 285 Asn Pro Glu Pro Thr Glu Glu Asp Ile AlaLys His Tyr Asn Val Lys 290 295 300 Thr Ala Ile Thr Arg Ala Pro Ala AspLeu Ala Ala Ala Leu Asn Val 305 310 315 320 Val Asp Asp Phe Cys Met LysThr Gly Leu Ser Phe Asp Glu Leu Leu 325 330 335 Asn Leu Thr Met Gln LysAsp Tyr Gln Ser Lys Ser Ser Glu Tyr Lys 340 345 350 Ser Arg Phe Val LysPhe Gly Gly Gly Glu His Val Pro Val Ser Thr 355 360 365 Tyr Gly Ala ValPhe Leu Thr Gly Thr Glu Glu Thr Pro Leu Trp Ala 370 375 380 Lys Gln TyrAsn Ser Ala Gly Ala Ala Thr Asp Thr Pro Val Leu Asn 385 390 395 400 PheThr Ala Asp Asn Val Ala Ala Leu Ala Gly Arg Ala Glu Lys Leu 405 410 415Val Arg Leu Ala Arg Ser Thr Gly Leu Ser Phe Glu Gln Leu 420 425 430 34340 DNA Paenibacillus apairius strain DB482 34 tatatttatt cacaccttggacttcctgat caaccgcggc gacagcttgt accggctgct 60 ggagcgggat actctgaccgaagccaagat gtattacatc caggccagcc aactgcttgg 120 tccccgcccc gatatccggatcaatcacag ttggcctaat ccgaccctgc aaagcgaagc 180 ggacgcggtg accgccgtaccgacgcgaag cgattcgcgg gcaacgccaa tcctcgcctt 240 gcgagcgctt ctgaaagcggaaaacgggca tttcctgccg ccttataatg atgaactgtt 300 agctttctgg gataaaatcgatctgcgttt atacaattta 340 35 565 DNA Paenibacillus apairius strain DB48235 gtctctatac tatcaaatgt atgacgccgc attgccgctc tgcttgatgg ccaaacaggc 60tttagagaaa gaaatcggca ctgataaaac gggtggagtt ttcaccctcc cggcctggaa 120tgatctgtat cagggattac tggcggggga ggcgctgctg ctcgagcttc agaagctgga 180gaatctgtgg ctggaggaag acaagcgcgg aatggaagcc gtaaaaacgg tatctttaga 240tacccttctc cgcaaagaaa cgccagagtc tagcttcgta gagctagtca aggaagttct 300ggacggaaag acgcctgacc ctgtaggcgg agtcggcgta cagctgcaaa acaatatttt 360cagcgcaacc cttgacctgt ccgttcttgg cttggatcgc tcttacaacc aagcggaaaa 420gacccgcagg atcaaaaatc tgtcggttac cttacccgcg cttttgggac cttaccagga 480tatagaagca accttatcgc taggcggcga gaccgttgcg ctttcccatg gcgtggatga 540cagcggcttg ttcatcacgg atctt 565 36 113 PRT Paenibacillus apairius strainDB482 36 Ile Phe Ile His Thr Leu Asp Phe Leu Ile Asn Arg Gly Asp Ser Leu1 5 10 15 Tyr Arg Leu Leu Glu Arg Asp Thr Leu Thr Glu Ala Lys Met TyrTyr 20 25 30 Ile Gln Ala Ser Gln Leu Leu Gly Pro Arg Pro Asp Ile Arg IleAsn 35 40 45 His Ser Trp Pro Asn Pro Thr Leu Gln Ser Glu Ala Asp Ala ValThr 50 55 60 Ala Val Pro Thr Arg Ser Asp Ser Arg Ala Thr Pro Ile Leu AlaLeu 65 70 75 80 Arg Ala Leu Leu Lys Ala Glu Asn Gly His Phe Leu Pro ProTyr Asn 85 90 95 Asp Glu Leu Leu Ala Phe Trp Asp Lys Ile Asp Leu Arg LeuTyr Asn 100 105 110 Leu 37 188 PRT Paenibacillus apairius strain DB48237 Ser Leu Tyr Tyr Gln Met Tyr Asp Ala Ala Leu Pro Leu Cys Leu Met 1 510 15 Ala Lys Gln Ala Leu Glu Lys Glu Ile Gly Thr Asp Lys Thr Gly Gly 2025 30 Val Phe Thr Leu Pro Ala Trp Asn Asp Leu Tyr Gln Gly Leu Leu Ala 3540 45 Gly Glu Ala Leu Leu Leu Glu Leu Gln Lys Leu Glu Asn Leu Trp Leu 5055 60 Glu Glu Asp Lys Arg Gly Met Glu Ala Val Lys Thr Val Ser Leu Asp 6570 75 80 Thr Leu Leu Arg Lys Glu Thr Pro Glu Ser Ser Phe Val Glu Leu Val85 90 95 Lys Glu Val Leu Asp Gly Lys Thr Pro Asp Pro Val Gly Gly Val Gly100 105 110 Val Gln Leu Gln Asn Asn Ile Phe Ser Ala Thr Leu Asp Leu SerVal 115 120 125 Leu Gly Leu Asp Arg Ser Tyr Asn Gln Ala Glu Lys Thr ArgArg Ile 130 135 140 Lys Asn Leu Ser Val Thr Leu Pro Ala Leu Leu Gly ProTyr Gln Asp 145 150 155 160 Ile Glu Ala Thr Leu Ser Leu Gly Gly Glu ThrVal Ala Leu Ser His 165 170 175 Gly Val Asp Asp Ser Gly Leu Phe Ile ThrAsp Leu 180 185 38 2091 DNA Paenibacillus apairius strain DB482 38caggcaacct catcggctgt ctgtggtgtg ccattcccga tcaacgtggt atcggacata 60cacacggtgg acgaaatcag cggcagcgcc aggattcaga agtatactta ccgcaatggc 120gtgtatgacc ggaccgataa ggaatttgcc gggttcggcc acattgacac atgggaagag 180gagcgggatt ccgagggaac ccttagcatc agcactcccc ccgtgctgac acggacctgg 240tatcataccg ggcaaaagca ggatgaggag cgtgccgtgc agcaatattg gcaaggcgac 300cctgccgctt ttcaggttaa acccgtccgg cttactcgat tcgatgcggc aacggcccag 360gatgtcccgc tagactctcc caataggcgg gaagagtatt ggctgtatcg ctcgttgcga 420gggatgccgc tgcgtaatga aatttttgct ggagatgttg tggggttgcc tccttatcag 480gtggagagct tacgttatca agtgcgcttg atgcagagca ccgattcgga atgtgttaca 540ttgcccatgc agttggagca gcttacgtac aactatgagc aaatcgcctc tgatccgcag 600tgttcacagc agatacagca atggttcgac gaatacggcg tggcggcaca gagtataacg 660atccaatatc cgcgccgggc acagccggag gacaatccgt accctcacac gctgccggat 720accagctgga gcagcagtta tgattcgcag caaatgctgc tgcggttaac aaggcaaagg 780caaaaagcgt accaccttgc agaccctgaa ggctggcgct tgaatatccc ccatcagaca 840cgcctggatt ctttcatcta ttctgctgac agcgtgcctg ccgaaggaat aagcgcagag 900ctgctggggg gtgacggcac gttacgatct ccggcgctgg aacaggctta tggcggccag 960tcagagatca tctatgcggg cgggggggaa ccggattcgc gagctctggt ccattacacc 1020agaagcgcga ttctcgatga agcctgtttg caagcctatg aaggcgtact gagcgatagc 1080caattgaact cgcttcttgc atcttccggc tatcaacgaa gcgcaagaat attgggttcc 1140ggcgatgaag cggatatttt tgttgcggaa caaggattta cccgttatgc ggatgaacag 1200aattttttcc gtattctggg acaacaatcc tctctcttga ccggggaaca agtattaaca 1260tgggatgata atttctgtgc ggtaacatcc atagaagacg cgcttggcaa tcaaattcag 1320attgcatatg attaccgctt tgtggaggct atccagatta ccgatgcgaa taacaatgtg 1380aatcaggtct ccctggatgc tctcggccgg gtcgtataca gccggacctg gggcacagag 1440gaagggatag agacgggctt ccgcccggag gcggaattct cgccgcccga gacaatggag 1500caggcgcttg ccctggcgtc tcccttgccg gttgcatcct gctgtgttta tgatgcgcat 1560agctggatgg gaacgataac tcttgggcag ctgtcagcgc ttgttccaga tagtgaaaag 1620caatggtcgt tcttgatagc caatcgcttg attatgccgg acggcaggat aagagcccgc 1680ggccgggccc catggtggct tcaacggcta ttgccgcctg ccgtggccaa gctgctgagc 1740gaggcggacc gtaagccgcc gcatacggta gttttggcag cagatcgcta cccggatgac 1800ccatcccagc aaattcaggc cagcgtcgtg tttagcgatg gctttgggcg tacgatacaa 1860accgctaaaa gagcagatac ccgatgggcg attacggaac ggattgacta tgacgaaacc 1920ggagccgtaa tccgaagctt tcagcctttt tatattgatg actggaatta tgtgggcaaa 1980gaggctgtca gcggctctat gtatgcaacg atctattact atgatgctct ggcacgccaa 2040ctaaggatgg tcaacgccaa aggatatgag aggagaactg ctttttaccc a 2091 39 697 PRTPaenibacillus apairius strain DB482 39 Gln Ala Thr Ser Ser Ala Val CysGly Val Pro Phe Pro Ile Asn Val 1 5 10 15 Val Ser Asp Ile His Thr ValAsp Glu Ile Ser Gly Ser Ala Arg Ile 20 25 30 Gln Lys Tyr Thr Tyr Arg AsnGly Val Tyr Asp Arg Thr Asp Lys Glu 35 40 45 Phe Ala Gly Phe Gly His IleAsp Thr Trp Glu Glu Glu Arg Asp Ser 50 55 60 Glu Gly Thr Leu Ser Ile SerThr Pro Pro Val Leu Thr Arg Thr Trp 65 70 75 80 Tyr His Thr Gly Gln LysGln Asp Glu Glu Arg Ala Val Gln Gln Tyr 85 90 95 Trp Gln Gly Asp Pro AlaAla Phe Gln Val Lys Pro Val Arg Leu Thr 100 105 110 Arg Phe Asp Ala AlaThr Ala Gln Asp Val Pro Leu Asp Ser Pro Asn 115 120 125 Arg Arg Glu GluTyr Trp Leu Tyr Arg Ser Leu Arg Gly Met Pro Leu 130 135 140 Arg Asn GluIle Phe Ala Gly Asp Val Val Gly Leu Pro Pro Tyr Gln 145 150 155 160 ValGlu Ser Leu Arg Tyr Gln Val Arg Leu Met Gln Ser Thr Asp Ser 165 170 175Glu Cys Val Thr Leu Pro Met Gln Leu Glu Gln Leu Thr Tyr Asn Tyr 180 185190 Glu Gln Ile Ala Ser Asp Pro Gln Cys Ser Gln Gln Ile Gln Gln Trp 195200 205 Phe Asp Glu Tyr Gly Val Ala Ala Gln Ser Ile Thr Ile Gln Tyr Pro210 215 220 Arg Arg Ala Gln Pro Glu Asp Asn Pro Tyr Pro His Thr Leu ProAsp 225 230 235 240 Thr Ser Trp Ser Ser Ser Tyr Asp Ser Gln Gln Met LeuLeu Arg Leu 245 250 255 Thr Arg Gln Arg Gln Lys Ala Tyr His Leu Ala AspPro Glu Gly Trp 260 265 270 Arg Leu Asn Ile Pro His Gln Thr Arg Leu AspSer Phe Ile Tyr Ser 275 280 285 Ala Asp Ser Val Pro Ala Glu Gly Ile SerAla Glu Leu Leu Gly Gly 290 295 300 Asp Gly Thr Leu Arg Ser Pro Ala LeuGlu Gln Ala Tyr Gly Gly Gln 305 310 315 320 Ser Glu Ile Ile Tyr Ala GlyGly Gly Glu Pro Asp Ser Arg Ala Leu 325 330 335 Val His Tyr Thr Arg SerAla Ile Leu Asp Glu Ala Cys Leu Gln Ala 340 345 350 Tyr Glu Gly Val LeuSer Asp Ser Gln Leu Asn Ser Leu Leu Ala Ser 355 360 365 Ser Gly Tyr GlnArg Ser Ala Arg Ile Leu Gly Ser Gly Asp Glu Ala 370 375 380 Asp Ile PheVal Ala Glu Gln Gly Phe Thr Arg Tyr Ala Asp Glu Gln 385 390 395 400 AsnPhe Phe Arg Ile Leu Gly Gln Gln Ser Ser Leu Leu Thr Gly Glu 405 410 415Gln Val Leu Thr Trp Asp Asp Asn Phe Cys Ala Val Thr Ser Ile Glu 420 425430 Asp Ala Leu Gly Asn Gln Ile Gln Ile Ala Tyr Asp Tyr Arg Phe Val 435440 445 Glu Ala Ile Gln Ile Thr Asp Ala Asn Asn Asn Val Asn Gln Val Ser450 455 460 Leu Asp Ala Leu Gly Arg Val Val Tyr Ser Arg Thr Trp Gly ThrGlu 465 470 475 480 Glu Gly Ile Glu Thr Gly Phe Arg Pro Glu Ala Glu PheSer Pro Pro 485 490 495 Glu Thr Met Glu Gln Ala Leu Ala Leu Ala Ser ProLeu Pro Val Ala 500 505 510 Ser Cys Cys Val Tyr Asp Ala His Ser Trp MetGly Thr Ile Thr Leu 515 520 525 Gly Gln Leu Ser Ala Leu Val Pro Asp SerGlu Lys Gln Trp Ser Phe 530 535 540 Leu Ile Ala Asn Arg Leu Ile Met ProAsp Gly Arg Ile Arg Ala Arg 545 550 555 560 Gly Arg Ala Pro Trp Trp LeuGln Arg Leu Leu Pro Pro Ala Val Ala 565 570 575 Lys Leu Leu Ser Glu AlaAsp Arg Lys Pro Pro His Thr Val Val Leu 580 585 590 Ala Ala Asp Arg TyrPro Asp Asp Pro Ser Gln Gln Ile Gln Ala Ser 595 600 605 Val Val Phe SerAsp Gly Phe Gly Arg Thr Ile Gln Thr Ala Lys Arg 610 615 620 Ala Asp ThrArg Trp Ala Ile Thr Glu Arg Ile Asp Tyr Asp Glu Thr 625 630 635 640 GlyAla Val Ile Arg Ser Phe Gln Pro Phe Tyr Ile Asp Asp Trp Asn 645 650 655Tyr Val Gly Lys Glu Ala Val Ser Gly Ser Met Tyr Ala Thr Ile Tyr 660 665670 Tyr Tyr Asp Ala Leu Ala Arg Gln Leu Arg Met Val Asn Ala Lys Gly 675680 685 Tyr Glu Arg Arg Thr Ala Phe Tyr Pro 690 695 40 858 DNAPaenibacillus apairius strain DB482 40 atcctgtcta tctgcaatga agcggagccggtccgttatt tccgcaatca ggccgtcgct 60 ccgaaaaggc agtatgctta cgatgccctgtatcagcttg tatccagctc ggggcgggaa 120 tccgacgcgc ttcgtcagca gacgtcgcttcctcccttga tcacgcctat tcctctcgac 180 gatagccaat acgtcaatta tgctgagagatacagctatg atcgggcggg caatctaatc 240 aagcttagcc atcatggggc aagtcaatatacaacgaatg tgcatgtgga caaaagttca 300 aaccggggga tttggcggca aggggaagacatcccggata tcgcggcttc ctttgacaga 360 gcaggcaatc aacaagattt attcccggggagacggttgg aatgggatac acgcaatcag 420 ttatgccgtg tccatatggt cgtgcgcgaaggcggcgata acgactggga gggctatctc 480 tatgacagct caggaatgcg catcgtaaaacattctaccc gcaagacaca gacgacaacg 540 caaacggata cgacgatcta tttgccgggcctggagcttc gcatccgcca aaccggggac 600 agggtcacgg aagcattgca ggtcattaccgtggatgagg gagcgggaca agtgagggtg 660 ctgcactggg aggatggaac cgagccgggcggcatagcca atgatcagta tcggtacagc 720 ctaaacgatc atcttggctc ctctttattggaagttgacg ggcaaagtca gatcattagc 780 aaggaagaat tttatcccta tggcggcacagcattgtgga cagcccggtc agaggtggag 840 gcaagctaca agaccacg 858 41 286 PRTPaenibacillus apairius strain DB482 41 Ile Leu Ser Ile Cys Asn Glu AlaGlu Pro Val Arg Tyr Phe Arg Asn 1 5 10 15 Gln Ala Val Ala Pro Lys ArgGln Tyr Ala Tyr Asp Ala Leu Tyr Gln 20 25 30 Leu Val Ser Ser Ser Gly ArgGlu Ser Asp Ala Leu Arg Gln Gln Thr 35 40 45 Ser Leu Pro Pro Leu Ile ThrPro Ile Pro Leu Asp Asp Ser Gln Tyr 50 55 60 Val Asn Tyr Ala Glu Arg TyrSer Tyr Asp Arg Ala Gly Asn Leu Ile 65 70 75 80 Lys Leu Ser His His GlyAla Ser Gln Tyr Thr Thr Asn Val His Val 85 90 95 Asp Lys Ser Ser Asn ArgGly Ile Trp Arg Gln Gly Glu Asp Ile Pro 100 105 110 Asp Ile Ala Ala SerPhe Asp Arg Ala Gly Asn Gln Gln Asp Leu Phe 115 120 125 Pro Gly Arg ArgLeu Glu Trp Asp Thr Arg Asn Gln Leu Cys Arg Val 130 135 140 His Met ValVal Arg Glu Gly Gly Asp Asn Asp Trp Glu Gly Tyr Leu 145 150 155 160 TyrAsp Ser Ser Gly Met Arg Ile Val Lys His Ser Thr Arg Lys Thr 165 170 175Gln Thr Thr Thr Gln Thr Asp Thr Thr Ile Tyr Leu Pro Gly Leu Glu 180 185190 Leu Arg Ile Arg Gln Thr Gly Asp Arg Val Thr Glu Ala Leu Gln Val 195200 205 Ile Thr Val Asp Glu Gly Ala Gly Gln Val Arg Val Leu His Trp Glu210 215 220 Asp Gly Thr Glu Pro Gly Gly Ile Ala Asn Asp Gln Tyr Arg TyrSer 225 230 235 240 Leu Asn Asp His Leu Gly Ser Ser Leu Leu Glu Val AspGly Gln Ser 245 250 255 Gln Ile Ile Ser Lys Glu Glu Phe Tyr Pro Tyr GlyGly Thr Ala Leu 260 265 270 Trp Thr Ala Arg Ser Glu Val Glu Ala Ser TyrLys Thr Thr 275 280 285 42 4434 DNA Photorhabdus strain W14 42atgatgcaga attcacaaac attcagtgtt accgagctgt cattacccaa aggcggcggc 60gctattaccg gtatgggtga agcattaaca ccagccgggc cggatggtat ggccgcctta 120tccctgccat tacccatttc cgccgggcgt ggttacgcac cctcgctcac tctgaattac 180aacagtggaa ccggtaacag cccatttggt ctcggttggg actgcggcgt catggcaatt 240cgtcgtcgca ccagtaccgg cgtaccgaat tacgatgaaa ccgatacttt tctggggccg 300gaaggtgaag tgttggtcgt agcattaaat gaggcaggtc aagctgatat ccgcagtgaa 360tcctcattgc agggcatcaa tttgggtgcg accttcaccg ttacctgtta tcgctcccgc 420ctagaaagcc actttaaccg gttggaatac tggcaacccc aaacaaccgg cgcaaccgat 480ttctggctga tatacagccc cgacggacag gtccatttac tgggcaaaaa tcctcaggca 540cgtatcagca atccactcaa tgttaaccaa acagcgcaat ggctgttgga agcctcgata 600tcatcccaca gcgaacagat ttattatcaa tatcgcgctg aagatgaagc aggttgtgaa 660accgacgagc tagcagccca ccccagcgca accgttcagc gctacctgca aacagtacat 720tacgggaacc tgaccgccag cgacgttttt cctacactaa acggagatga cccacttaaa 780tctggctgga tgttctgttt agtatttgac tacggtgagc gcaaaaacag cttatctgaa 840atgccgctgt ttaaagccac aggcaattgg ctttgccgaa aagaccgttt ttcccgttat 900gagtacggtt ttgaattgcg tactcgccgc ttatgccgcc aaatactgat gtttcaccgt 960ctacaaaccc tatctggtca ggcaaagggg gatgatgaac ctgcgctagt gtcgcgtctg 1020atactggatt atgacgaaaa cgcgatggtc agtacgctcg tttctgtccg ccgggtaggc 1080catgaggaca acaacacggt taccgcgctg ccaccactgg aactggccta tcagcctttt 1140gagccagaac aaaccgcact ctggcaatca atggatgtac tggcaaattt caacaccatt 1200cagcgctggc aactgcttga cctgaaagga gaaggcgtgc ccggcattct ctatcaggat 1260agaaatggct ggtggtatcg atctgcccaa cgtcaggccg gggaagagat gaatgcggtc 1320acctggggga aaatgcaact ccttcccatc acaccagctg tgcaggataa cgcctcactg 1380atggatatta acggtgacgg gcaactggac tgggtgatta ccgggccggg gctaaggggc 1440tatcacagcc aacacccgga tggcagttgg acgcgtttta cgccattaca tgccctgccg 1500atagaatatt ctcatcctcg cgctcaactt gccgatttaa tgggagccgg gctgtccgat 1560ttagtgctaa ttggtcccaa aagtgtgcgc ttatatgtca ataaccgtga tggttttacc 1620gaagggcggg atgtggtgca atccggtgat atcaccctgc cgctaccggg cgccgatgcc 1680cgtaagttag tggcatttag tgacgtactg ggttcaggcc aagcacatct ggttgaagtt 1740agtgcaactc aagtcacctg ctggccgaat ctggggcatg gccgttttgg tcagccaatc 1800gtattgccgg gattcagcca atctgccgcc agttttaatc ctgatcgagt tcatctggcc 1860gatttggatg ggagcggccc tgccgatttg atttatgttc atgctgaccg tctggatatt 1920ttcagcaatg aaagtggcaa cggttttgca aaaccattca cactctcttt tcctgacggc 1980ctgcgttttg atgatacctg ccagttgcaa gtagccgatg tacaagggtt aggcgttgtc 2040agcctgatcc taagcgtacc gcatatggcg ccacatcatt ggcgctgcga tctgaccaac 2100gcgaaaccgt ggttactcag tgaaacgaac aacaatatgg gggccaatca caccttgcat 2160taccgtagct ctgtccagtt ctggctggat gaaaaagctg cggcattggc taccggacaa 2220acaccggtct gttacctgcc cttcccggtc catacccttt ggcaaacaga aaccgaggat 2280gaaatcagcg gcaataagtt agtgaccacg ttacgttatg ctcacggcgc ttgggatgga 2340cgtgaacggg aatttcgtgg ctttggttat gttgagcaga cagacagcca tcaactcgct 2400caaggcaatg cgccggaacg tacaccaccg gcactcacca aaagctggta tgccaccgga 2460ttacctgcgg tagataatgc gttatccgcc gggtattggc gtggcgataa gcaagctttc 2520gccggtttta cgccacgttt tactctctgg aaagagggca aagatgttcc actgacaccg 2580gaagatgacc ataatctata ctggttaaac cgggcgctaa aaggtcagcc actgcgtagt 2640gaactctacg ggctggatgg cagcgcacag caacagatcc cctatacagt gactgaatcc 2700cgtccacagg tgcgccaatt acaagatggc gccaccgttt ccccggtgct ctgggcctca 2760gtcgtggaaa gccgtagtta tcactacgaa cgtattatca gtgatcccca gtgcaatcag 2820gatatcacgt tgtccagtga cctattcggg caaccactga aacaggtttc cgtacaatat 2880ccccgccgca acaaaccaac aaccaatccg tatcccgata ccctaccgga tacgctgttt 2940gccagcagtt atgacgatca acaacagcta ttgcgattaa cctgccgaca atccagttgg 3000caccatctta ttggtaatga gctaagagtg ttgggattac cggatggcac acgcagtgat 3060gcctttactt acgatgccaa acaggtacct gtcgatggct taaatctgga aaccctgtgt 3120gctgaaaata gcctgattgc cgatgataaa cctcgcgaat acctcaatca gcaacgaacg 3180ttctataccg acgggaaaaa ccaaacaccg ctgaaaacac cgacacgaca agcgttaatc 3240gcctttaccg aaacggcggt attaacggaa tctctgttat ccgcgtttga tggcggtatt 3300acgccagacg aattaccggg aatactgaca caggccggat accaacaaga gccttatctg 3360tttccacgca ccggcgaaaa caaagtttgg gtagcgcgtc aaggctatac cgattacggg 3420acggaagcac aattttggcg tcctgtcgca caacgtaaca gcctgttaac cgggaaaatg 3480acgttaaaat gggatactca ctattgtgtc atcacccaaa cccaagatgc tgccggcctc 3540accgtctcag ccaattatga ctggcgtttt ctcacaccaa cgcaactgac tgacatcaac 3600gataatgtgc atctcatcac cttggatgct ctgggacgcc ctgtcacgca acgtttctgg 3660gggatcgaaa gcggtgtggc aacaggttac tcttcatcag aagaaaaacc attctctcca 3720ccaaacgata tcgataccgc tattaatcta accggaccac tccctgtcgc acagtgtctg 3780gtctatgcac cggacagttg gatgccacta ttcagtcaag aaaccttcaa cacattaacg 3840caggaagagc aggagacgct gcgtgattca cgtattatca cggaagattg gcgtatttgc 3900gcactgactc gccgccgttg gctacaaagt caaaagatca gtacaccatt agttaaactg 3960ttaaccaaca gcattggttt acctccccat aaccttacgc tgaccacaga ccgttatgac 4020cgcgactctg agcagcaaat tcgccaacaa gtcgcattta gtgatggttt tggccgtctg 4080ctacaagcgt ctgtacgaca tgaggcaggc gaagcctggc aacgtaacca agacggttct 4140ctggtgacaa aagtggagaa taccaaaacg cgttgggcgg tcacgggacg caccgaatat 4200gataataaag ggcaaacgat acgcacttat cagccctatt tcctcaacga ctggcgatat 4260gtcagtgatg acagcgccag aaaagaagcc tatgcggata ctcatattta tgatccaatt 4320gggcgagaaa tccgggttat tactgcaaaa ggctggctgc gccaaagcca atatttcccg 4380tggtttaccg tgagtgagga tgagaatgat acggccgctg atgcgctggt gtaa 4434 43 4425DNA Photorhabdus strain W14 43 atgcaaaatt cacaagattt tagtattacggaactgtcac tgcccaaagg ggggggcgct 60 atcacgggaa tgggtgaagc attaacccccactggaccgg atggtatggc cgcgctatct 120 ctaccattgc ctatttctgc cgggcgcggttatgctcccg cattcactct gaattacaac 180 agcggcgccg gtaacagtcc atttggtctgggttgggatt gcaacgttat gactatccgc 240 cgccgcaccc attttggcgt cccccattatgacgaaaccg ataccttttt ggggccagaa 300 ggcgaagtgc tggtggtagc ggatcaacctcgcgacgaat ccacattaca gggtatcaat 360 ttaggcgcca cctttaccgt taccggctaccgttcccgtc tggaaagcca tttcagccga 420 ttggaatatt ggcaacccaa aacaacaggtaaaacagatt tttggttgat atatagccca 480 gatgggcagg tgcatctact gggtaaatcaccgcaagcgc ggatcagcaa cccatcccaa 540 acgacacaaa cagcacaatg gctgctggaagcctctgtat catcacgtgg cgaacaaatt 600 tattatcaat atcgcgccga agatgacacaggttgcgaag cagatgaaat tacgcaccat 660 ttacaggcta cagcgcaacg ttatttacacatcgtgtatt acggcaaccg tacagccagc 720 gaaacattac ccggtctgga tggcagcgccccatcacaag cagactggtt gttctatctg 780 gtatttgatt acggcgaacg cagtaacaacctgaaaacgc caccagcatt ttcgactaca 840 ggtagctggc tttgccgtca ggaccgtttttcccgttatg aatatggctt tgagattcgt 900 acccgccgct tatgccgtca ggtattgatgtaccatcacc tgcaagcact ggatagtaag 960 ataacagaac acaacggacc aacgctggtttcacgcctga tactcaatta cgacgaaagc 1020 gcgatagcca gcacgctagt attcgttcgccgagtgggac acgagcaaga tggtaatgtc 1080 gtcaccctgc cgccattaga attggcatatcaggattttt caccgcgaca tcacgctcac 1140 tggcaaccaa tggatgtact ggcaaacttcaatgccattc agcgctggca gctagtcgat 1200 ctaaaaggcg aaggattacc cggcctgttatatcaggata aaggcgcttg gtggtaccgc 1260 tccgcacagc gtctgggcga aattggctcagatgccgtca cttgggaaaa gatgcaacct 1320 ttatcggtta ttccttcttt gcaaagtaatgcctcgttgg tggatatcaa tggagacggc 1380 caacttgact gggttatcac cggaccgggattacggggat atcatagtca acgcccggat 1440 ggcagttgga cacgttttac cccactcaacgctctgccgg tggaatacac ccatccacgc 1500 gcgcaactcg cagatttaat gggagccgggctatccgatt tggtgctgat cggccctaag 1560 agcgtgcgtt tatatgccaa tacccgcgacggctttgcca aaggaaaaga tgtggtgcaa 1620 tccggtgata tcacactgcc ggtgccgggcgccgatccac gtaagttggt ggcgtttagt 1680 gatgtattgg gttcaggtca agcccatctggttgaagtaa gcgcgactaa agtcacctgc 1740 tggcctaatc tggggcgcgg acgttttggtcaacccatta ccttaccggg attcagccag 1800 ccagcaaccg agtttaaccc ggctcaagtttatctggccg atctggatgg cagcggtcca 1860 acggatctga tttatgttca tacaaaccgtctggatatct tcctgaacaa aagtggcaat 1920 ggctttgctg aaccagtgac attacgcttcccggaaggtc tgcgttttga tcatacctgt 1980 cagttacaaa tggccgatgt acaaggattaggcgtcgcca gcctgatact gagcgtgccg 2040 catatgtctc cccatcactg gcgctgcgatctgaccaaca tgaagccgtg gttactcaat 2100 gaaatgaaca acaatatggg ggtccatcacaccttgcgtt accgcagttc ctcccaattc 2160 tggctggatg aaaaagccgc ggcgctgactaccggacaaa caccggtttg ctatctcccc 2220 ttcccgatcc acaccctatg gcaaacggaaacagaagatg aaatcagcgg caacaaatta 2280 gtcacaacac ttcgttatgc tcgtggcgcatgggacggac gcgagcggga atttcgcgga 2340 tttggttatg tagagcagac agacagccatcaactggctc aaggcaacgc gccagaacgt 2400 acgccaccgg cgctgaccaa aaactggtatgccaccggac tgccggtgat agataacgca 2460 ttatcaaccg agtattggcg tgatgatcaggcttttgccg gtttctcacc gcgctttacg 2520 acttggcaag ataacaaaga tgtcccgttaacaccggaag atgataacag tcgttactgg 2580 ttcaaccgcg cgttgaaagg tcaactgctacgtagtgaac tgtacggatt ggacgatagt 2640 acaaataaac acgttcccta tactgtcactgaatttcgtt cacaggtacg tcgattacag 2700 cataccgaca gccgataccc tgtactttggtcatctgtag ttgaaagccg caactatcac 2760 tacgaacgta tcgccagcga cccgcaatgcagtcaaaata ttacgctatc cagtgatcga 2820 tttggtcagc cgctaaaaca gctttcggtacagtacccgc gccgccagca gccagcaatc 2880 aatctgtatc ctgatacatt gcctgataagttgttagcca acagctatga tgaccaacaa 2940 cgccaattac ggctcaccta tcaacaatccagttggcatc acctgaccaa caataccgtt 3000 cgagtattgg gattaccgga tagtacccgcagtgatatct ttacttatgg cgctgaaaat 3060 gtgcctgctg gtggtttaaa tctggaacttctgagtgata aaaatagcct gatcgcggac 3120 gataaaccac gtgaatacct cggtcagcaaaaaaccgctt ataccgatgg acaaaataca 3180 acgccgttgc aaacaccaac acggcaagccctgattgcct ttaccgaaac aacggtattc 3240 aaccagtcca cattatcagc gtttaacggaagcatcccgt ccgataaatt atcaacgacg 3300 ctggagcaag ctggatatca gcaaacaaattatctattcc ctcgcactgg agaagataaa 3360 gtttgggtag cccatcacgg ctataccgattatggtacag cggcacagtt ctggcgcccg 3420 caaaaacaga gcaacaccca actcaccggtaaaatcaccc tcatctggga tgcaaactat 3480 tgcgttgtgg tacaaacccg ggatgctgctggactgacaa cctcagccaa atatgactgg 3540 cgttttctga ccccggtgca actcaccgatatcaatgaca atcagcacct tatcacactg 3600 gatgcattgg gccgaccaat cacattgcgcttttggggaa ctgaaaacgg caagatgaca 3660 ggttattcct caccggaaaa agcatcattttctccaccat ccgatgttaa tgccgctatt 3720 gagttaaaaa aaccgctccc tgtagcacagtgtcaggtct acgcaccaga aagctggatg 3780 ccagtattaa gtcagaaaac cttcaatcgactggcagaac aagattggca aaagttatat 3840 aacgcccgaa tcatcaccga agatggacgtatctgcacac tggcttatcg ccgctgggta 3900 caaagccaaa aggcaatccc tcaactcattagcctgttaa acaacggacc ccgtttacct 3960 cctcacagcc tgacattgac gacggatcgttatgatcacg atcctgagca acagatccgt 4020 caacaggtgg tattcagtga tggctttggccgcttgctgc aagccgctgc ccgacatgag 4080 gcaggcatgg cccggcaacg caatgaagacggctctttga ttataaatgt ccagcatact 4140 gagaaccgtt gggcagtgac tggacgaacggaatatgaca ataaggggca accgatacgt 4200 acctatcagc cctatttcct caatgactggcgatacgtca gcaatgatag tgcccggcag 4260 gaaaaagaag cttatgcaga tacccatgtctatgatccca taggtcgaga aatcaaggtt 4320 atcaccgcaa aaggttggtt ccgtcgaaccttgttcactc cctggtttac tgtcaatgaa 4380 gatgaaaatg acacagccgc tgaggtgaagaaggtaaaga tgtaa 4425 44 3132 DNA Photorhabdus strain W14 44 atgagcccgtctgagactac tctttatact caaaccccaa cagtcagcgt gttagataat 60 cgcggtctgtccattcgtga tattggtttt caccgtattg taatcggggg ggatactgac 120 acccgcgtcacccgtcacca gtatgatgcc cgtggacacc tgaactacag tattgaccca 180 cgcttgtatgatgcaaagca ggctgataac tcagtaaagc ctaattttgt ctggcagcat 240 gatctggccggtcatgccct gcggacagag agtgtcgatg ctggtcgtac tgttgcattg 300 aatgatattgaaggtcgttc ggtaatgaca atgaatgcga ccggtgttcg tcagacccgt 360 cgctatgaaggcaacacctt gcccggtcgc ttgttatctg tgagcgagca agttttcaac 420 caagagagtgctaaagtgac agagcgcttt atctgggctg ggaatacaac ctcggagaaa 480 gagtataacctctccggtct gtgtatacgc cactacgaca cagcgggagt gacccggttg 540 atgagtcagtcactggcggg cgccatgcta tcccaatctc accaattgct ggcggaaggg 600 caggaggctaactggagcgg tgacgacgaa actgtctggc agggaatgct ggcaagtgag 660 gtctatacgacacaaagtac cactaatgcc atcggggctt tactgaccca aaccgatgcg 720 aaaggcaatattcagcgtct ggcttatgac attgccggtc agttaaaagg gagttggttg 780 acggtgaaaggccagagtga acaggtgatt gttaagtccc tgagctggtc agccgcaggt 840 cataaattgcgtgaagagca cggtaacggc gtggttacgg agtacagtta tgagccggaa 900 actcaacgtctgataggtat caccacccgg cgtgccgaag ggagtcaatc aggagccaga 960 gtattgcaggatctacgcta taagtatgat ccggtgggga atgttatcag tatccataat 1020 gatgccgaagctacccgctt ttggcgtaat cagaaagtgg agccggagaa tcgctatgtt 1080 tatgattctctgtatcagct tatgagtgcg acagggcgtg aaatggctaa tatcggtcag 1140 caaagcaaccaacttccctc acccgttata cctgttccta ctgacgacag cacttatacc 1200 aattaccttcgtacctatac ttatgaccgt ggcggtaatt tggttcaaat ccgacacagt 1260 tcacccgcgactcaaaatag ttacaccaca gatatcaccg tttcaagccg cagtaaccgg 1320 gcggtattgagtacattaac gacagatcca acccgagtgg atgcgctatt tgattccggc 1380 ggtcatcagaagatgttaat accggggcaa aatctggatt ggaatattcg gggtgaattg 1440 caacgagtcacaccggtgag ccgtgaaaat agcagtgaca gtgaatggta tcgctatagc 1500 agtgatggcatgcggctgct aaaagtgagt gaacagcaga cgggcaacag tactcaagta 1560 caacgggtgacttatctgcc gggattagag ctacggacaa ctggggttgc agataaaaca 1620 accgaagatttgcaggtgat tacggtaggt gaagcgggtc gcgcacaggt aagggtattg 1680 cactgggaaagtggtaagcc gacagatatt gacaacaatc aggtgcgcta cagctacgat 1740 aatctgcttggctccagcca gcttgaactg gatagcgaag ggcagattct cagtcaggaa 1800 gagtattatccgtatggcgg tacggcgata tgggcggcga gaaatcagac agaagccagc 1860 tacaaatttattcgttactc cggtaaagag cgggatgcca ctggattgta ttattacggc 1920 taccgttattatcaaccttg ggtgggtcga tggttgagtg ctgatccggc gggaaccgtg 1980 gatgggctgaatttgtaccg aatggtgagg aataacccca tcacattgac tgaccatgac 2040 ggattagcaccgtctccaaa tagaaatcga aatacatttt ggtttgcttc atttttgttt 2100 cgtaaacctgatgagggaat gtccgcgtca atgagacggg gacaaaaaat tggcagagcc 2160 attgccggcgggattgcgat tggcggtctt gcggctacca ttgccgctac ggctggcgcg 2220 gctatccccgtcattctggg ggttgcggcc gtaggcgcgg ggattggcgc gttgatggga 2280 tataacgtcggtagcctgct ggaaaaaggc ggggcattac ttgctcgact cgtacagggg 2340 aaatcgacgttagtacagtc ggcggctggc gcggctgccg gagcgagttc agccgcggct 2400 tatggcgcacgggcacaagg tgtcggtgtt gcatcagccg ccggggcggt aacaggggct 2460 gtgggatcatggataaataa tgctgatcgg gggattggcg gcgctattgg ggccgggagt 2520 gcggtaggcaccattgatac tatgttaggg actgcctcta cccttaccca tgaagtcggg 2580 gcagcggcgggtggggcggc gggtgggatg atcaccggta cgcaagggag tactcgggca 2640 ggtatccatgccggtattgg cacctattat ggctcctgga ttggttttgg tttagatgtc 2700 gctagtaaccccgccggaca tttagcgaat tacgcagtgg gttatgccgc tggtttgggt 2760 gctgaaatggctgtcaacag aataatgggt ggtggatttt tgagtaggct cttaggccgg 2820 gttgtcagcccatatgccgc cggtttagcc agacaattag tacatttcag tgtcgccaga 2880 cctgtctttgagccgatatt tagtgttctc ggcgggcttg tcggtggtat tggaactggc 2940 ctgcacagagtgatgggaag agagagttgg atttccagag cgttaagtgc tgccggtagt 3000 ggtatagatcatgtcgctgg catgattggt aatcagatca gaggcagggt cttgaccaca 3060 accgggatcgctaatgcgat agactatggc accagtgctg tgggagccgc acgacgagtt 3120 ttttctttgtaa 3132 45 2748 DNA Photorhabdus strain W14 45 atgagcagtt acaattctgcaattgaccaa aagaccccct cgattaaggt attagataac 60 aggaaattaa atgtacgtactttagaatat ctacgcactc aagctgacga aaacagtgat 120 gaattaatta cgttctatgagttcaatatt ccgggatttc aggtaaaaag caccgatcct 180 cgtaaaaata aaaaccagagcggcccaaat ttcattcgtg tctttaatct tgccggtcaa 240 gttttacgtg aagaaagtgttgatgccggt cggactatta ccctcaatga tattgaaagt 300 cgcccggtgt tgatcatcaatgcaaccggt gtccgccaaa accatcgtta tgaagataac 360 acccttcccg gtcgtctgctcgctatcacc gaacaagtac aggcaggaga gaaaacgacc 420 gaacgtctta tctgggccggcaatacgccg caagaaaaag attacaacct cgccggtcag 480 tgtgtccgcc attacgataccgcgggactt actcaactca atagcctttc tctggctggc 540 gtcgtgctat cacaatctcagcaactactc gtcgatgata aaaatgctga ctggacaggt 600 gaagaccaaa gcctctggcagcaaaaactg agcagtgatg tctataccac ccaaaataaa 660 gccgatgcca ccggggctttattgacccag accgatgcca aaggcaacat ccagcgtctg 720 gcctacgacg tagccgggcagctaaaaggc tgttggttga cactcaaagg tcaggccgag 780 caagtgatta tcaaatcgctgacctactcc gccgccggac aaaaattacg cgaagagcac 840 ggtaacgggg ttatcactgaatacagctat gaaccagaaa cccaacggct tatcggtatt 900 gccacccgcc gtccgtcagacgccaaagtg ttgcaagact tacgctatca atatgacccg 960 gtaggcaatg tgatcaatatccgtaatgat gcggaagcca cccgcttttg gcgcaatcag 1020 aaagtggtcc cggagaatagctatacctac gactccctgt atcagcttat cagtgccacc 1080 gggcgggaaa tggctaatataggtcagcaa aataaccaac tgccctcccc tgcgctacct 1140 tctgacaaca atacctacactaactatact cgcagctaca gctatgatca cagtggtaat 1200 ctgacgcaaa ttcggcacagctcgccagct acccagaaca actacaccgt ggctatcacc 1260 ctctcaaacc gcagcaatcggggtgttctc agtacgctaa ccaccgatcc aaatcaagtg 1320 gatacgttgt ttgatgccggtggtcaccaa accagtttat tacccggaca gacacttatc 1380 tggacaccac gaggagagttaaagcaggtt aataatggcc cgggaaatga gtggtaccgc 1440 tacgacagca acggcatgagacaactgaaa gtgagtgaac agccaaccca gaatactacg 1500 cagcaacaac gggtaatctatttgccggga ctggagctac gcacaaccca gagcaacgcc 1560 acaacaacgg aagagttacacgttatcaca ctcggtgaag ccggtcgcgc acaggtacgg 1620 gtgttgcact gggagagcggtaagccagaa gatgtcaaca ataatcaact acgttacagc 1680 tacgataatc tgatcggctccagccagctt gaactggaca accaaggaca aattatcagc 1740 gaggaagagt attatccatttggcgggaca gcgctgtggg cagcaaacag ccaaacagaa 1800 gccagctata aaacgattcgctattccggc aaagaacgag atgccaccgg gttgtattat 1860 tacggttatc gttattaccaaccgtgggcg ggcagatggt taagcgcgga cccggcagga 1920 accattgatg ggctgaatctataccgaatg gtaagaaata atcctgtgag tttacaagat 1980 gaaaatggat tagcgccagaaaaagggaaa tataccaaag aggtaaattt ctttgatgaa 2040 ttaaaattca aattggcagccaaaagttca catgttgtca aatggaacga gaaagagagc 2100 agttatacaa aaaataaatcattgaaagtg gttcgtgtcg gtgattccga tccgtcgggt 2160 tatttgctaa gccacgaagagttactaaaa ggtatagaaa aaagtcaaat catatatagc 2220 cgacttgaag aaaacagctccctttcagaa aaatcaaaaa cgaatctttc tttaggatct 2280 gaaatatccg gttatatggcaagaaccata caagatacga tatcagaata tgccgaagag 2340 cataaatata gaagtaatcaccctgatttt tattcagaaa ccgatttctt tgcgttaatg 2400 gataaaagtg aaaaaaatgattattccggt gaaagaaaaa tttatgcggc aatggaggtt 2460 aaggtttatc atgatttaaaaaataaacaa tcagaattac atgtcaacta tgcattggcc 2520 catccctata cgcaattgagtaatgaagaa agagcgctgt tgcaagaaac agaacccgct 2580 attgcaatag atagagaatataatttcaaa ggtgttggca aattcctgac aatgaaagca 2640 attaaaaaat cattgaaaggacataaaatt aataggatat caacagaggc tattaatatt 2700 cgctctgcgg ctatcgctgagaatttagga atgcggagaa cttcataa 2748 46 2883 DNA Photorhabdus strain W1446 atgaaaaaca ttgatcccaa actttatcaa aaaaccccta ctgtcagcgt ttacgataac 60cgtggtctga taatccgtaa catcgatttt catcgtacta ccgcaaatgg tgatcccgat 120acccgtatta cccgccatca atacgatatt cacggacacc taaatcaaag catcgatccg 180cgcctatatg aagccaagca aaccaacaat acgatcaaac ccaattttct ttggcagtat 240gatttgaccg gtaatcccct atgtacagag agcattgatg caggtcgcac tgtcaccttg 300aatgatattg aaggccgtcc gctactaacg gtgactgcaa caggggttat acaaactcga 360caatatgaaa cttcttccct gcccggtcgt ctgttatctg ttgccgaaca aacacccgag 420gaaaaaacat cccgtatcac cgaacgcctg atttgggctg gcaataccga agcagagaaa 480gaccataacc ttgccggcca gtgcgtgcgt cactatgaca cggcgggagt tacccggtta 540gagagtttat cactgaccgg tactgtttta tctcaatcca gccaactatt gatcgacact 600caagaggcaa actggacagg tgataacgaa accgtctggc aaaacatgct ggctgatgac 660atctacacaa ccctgagcac cttcgatgcc accggtgctt tactgactca gaccgatgcg 720aaagggaaca ttcagagact ggcttatgat gtggccgggc agctaaacgg gagctggcta 780acactcaaag gccagacgga acaagtgatt atcaaatccc tgacctactc cgccgccgga 840caaaaattac gtgaggaaca cggcaatgat gttatcaccg aatacagtta tgaaccggaa 900acccaacggc tgatcggtat caaaacccgc cgtccgtcag acactaaagt gctacaagac 960ctgcgctatg aatatgaccc ggtaggcaat gtcatcagca tccgtaatga cgcggaagcc 1020acccgctttt ggcacaatca gaaagtgatg ccggaaaaca cttataccta cgattccctg 1080tatcagctta tcagcgccac cgggcgcgaa atggcgaata taggtcaaca aagtcaccaa 1140tttccctcac ccgctctacc ttctgataac aacacctata ccaactatac ccgtacttat 1200acttatgacc gtggcggcaa tctgaccaaa atccagcaca gttcaccggc gacgcaaaac 1260aactacacca ccaatatcac ggtttcaaat cgcagcaacc gcgcagtact cagcacattg 1320accgaagatc cggcgcaagt agatgctttg tttgatgcag gcggacatca gaacaccttg 1380atatcaggac aaaacctgaa ctggaatact cgtggtgaac tgcaacaagt aacactggtt 1440aaacgggaca agggcgccaa tgatgatcgg gaatggtatc gttatagcgg tgacggaaga 1500aggatgttaa aaatcaatga acagcaggcc agcaacaacg ctcaaacaca acgtgtgact 1560tatttgccga acttagaact tcgtctaaca caaaacagca cggccacaac cgaagatttg 1620caagttatca ccgtaggcga agcgggccgg gcacaggtac gagtattaca ttgggagagc 1680ggtaaaccgg aagatatcga caataatcag ttgcgttata gttacgataa tcttatcggt 1740tccagtcaac ttgaattaga tagcgaagga caaattatca gtgaagaaga atattatccc 1800tatggtggaa cagcattatg ggccgccagg aatcagacag aagccagtta taaaactatc 1860cgttattcag gcaaagagcg ggatgccacc gggctatatt actacggcta tcggtattac 1920caaccgtgga taggacggtg gttaagctcc gatccggcag gaacaatcga tgggctgaat 1980ttatatcgga tggtgaggaa taatccagtt accctccttg atcctgatgg attaatgcca 2040acaattgcag aacgcatagc agcactaaaa aaaaataaag taacagactc agcgccttcg 2100ccagcaaatg ccacaaacgt agcgataaac atccgcccgc ctgtagcacc aaaacctagc 2160ttaccgaaag catcaacgag tagccaacca accacacacc ctatcggagc tgcaaacata 2220aaaccaacga cgtctgggtc atctattgtt gctccattga gtccagtagg aaataaatct 2280acttctgaaa tctctctgcc agaaagcgct caaagcagtt cttcaagcac tacctcgaca 2340aatctacaga aaaaatcatt tactttatat agagcagata acagatcctt tgaagaaatg 2400caaagtaaat tccctgaagg atttaaagcc tggactcctc tagacactaa gatggcaagg 2460caatttgcta gtatctttat tggtcagaaa gatacatcta atttacctaa agaaacagtc 2520aagaacataa gcacatgggg agcaaagcca aaactaaaag atctctcaaa ttacataaaa 2580tataccaagg acaaatctac agtatgggtt tctactgcaa ttaatactga agcaggtgga 2640caaagctcag gggctccact ccataaaatt gatatggatc tctacgagtt tgccattgat 2700ggacaaaaac taaatccact accggagggt agaactaaaa acatggtacc ttccctttta 2760ctcgacaccc cacaaataga gacatcatcc atcattgcac ttaatcatgg accggtaaat 2820gatgcagaaa tttcatttct gacaacaatt ccgcttaaaa atgtaaaacc tcataagaga 2880taa 2883 47 2850 DNA Photorhabdus strain W14 47 atgaaaaaca ttgacccaaaactttatcaa catacgccca ccgttaacgt ctacgataac 60 cgtggcctga ccattcgtaacatcgacttt caccgtgacg tcgcgggagg cgatacagat 120 actcgtatta cccgccaccaatatgatacc cgaggacact tgagccaaag cattgatcca 180 cggctgtatg acgccaaacaaaccaataac tcgacaaacc ccaacttcct ctggcaatac 240 aatctcaccg gcgacactttgcggacagaa agtgtcgatg ccggccgtac cgtagccctc 300 aatgatattg aaggccgtcaagtgttgatt gtaaccgcaa ccggcgccat tcagacccga 360 caatatgaag ccaataccctgcccggtcgt ctattatccg taagtgaaca agcccccgga 420 gaacagactc cccgcgttactgagcatttt atttgggctg gtaatacaca ggcggagaaa 480 gatcataatc ttgccggccagtatgtgcgc cactacgaca cagcaggagt gacgcaactg 540 gaaagcctgt cattgacagaaaacatctta tctcaatccc gtcagttatt agccgacggt 600 caggaagcag actggacaggtaacgatgaa accctctggc agaccaaact caatagcgaa 660 acttacacga cacaaagcacctttgatgct accggcgctt tgctgaccca aaccgatgca 720 aaaggcaaca tgcaacgtctggcttacaac gtggcaggac aattacaagg tagctggctg 780 acattgaaaa accaaagtgagcaagtcatt gtcaaatccc tgacctattc cgccgcaggc 840 cagaaattgc gtgaagaacacggtaatggc gttatcactg aatacagcta tgaaccggaa 900 actctacgat tgatcggtaccactactcgc cgtcaatcag atagcaaggt gttacaagat 960 ctacgctatg aacatgatcctgtagggaat attattagtg tccgtaatga tgcagaagcc 1020 acccgcttct ggcgcaatcagaaaatagtc cctgaaaata cctacaccta cgattccctg 1080 tatcagctta tcagtgcaacaggacgtgag atggctaaca tcggccagca aagcaaccaa 1140 cttccttcgc caatcatccctcttcctact gatgaaaact catataccaa ctatactcgc 1200 agctataatt acgatcgcggcggcaatttg gttcaaatcc ggcacagttc ccccgccgcc 1260 caaaataact acaccacagatatcaccgtt tcgaatcgca gtaaccgggc agtgctgagt 1320 tcgctaacct cagacccaacacaggtggag gcactgtttg atgccggcgg acatcaaaca 1380 aaattgttac cggggcaagagctgagttgg aatacacgag gtgaactaaa acaggtaacg 1440 ccagtcagtc gcgagagcgccagcgatcgg gaatggtatc gttacggcaa cgacggcatg 1500 cgacggttaa aagtcagtgagcaacagact ggcaacagca cgcagcagca acgagtaact 1560 tatcttcccg atctggagctacgtacaaca caaaatggga ctactacatc agaagacctg 1620 catgctatta ccgtgggagcagcaggccac gcacaagtgc gagttctaca ctgggaaact 1680 acgccaccag ccggtatcaataacaatcag cttcgctata gctatgataa tttgattggt 1740 tccagtcaac ttgaactggataacgcagga caaattatca gtcaggaaga gtattatcca 1800 tttggcggca cagcattatgggcagcaaga aaccaaatag aagccagcta caaaatcctc 1860 cgttactcag gtaaagaacgcgatgctacc gggctctatt attacggcta ccgctattat 1920 cagccgtggg ttggtaggtggttaagcgcc gatccggctg gaacaatcga tggactgaat 1980 ctataccgga tggtgagaaataatccgtca acactggttg atatttctgg gcttgcacct 2040 acgaaataca atattcccggatttgacttt gatgtagaaa tagatgagca aaaaagatct 2100 aaattaaaac caacgttgataagaatcaaa gatgaatttt tacattatgg tcctgtagat 2160 aagctgttag aagaaaaaaaacccggcctc aatgtaccag aggagctatt tgatagaggt 2220 ccatccgaga atggagtgtcaacattaact ttcaaaaaag acctaccgat aagttgtatt 2280 agcaacacag aatatacccttgatatctta tacaacaaac atgagactaa accattccct 2340 tacgaaaacg aagcaacagttggcgcagat ctgggagtaa taatgtccgt ggagtttgga 2400 aataaatcaa taggtaatgcctctgacgaa gatttaaaag aagaacatct cccattagga 2460 aaatccacaa tggataaaacagacctgcca gatttaaaac aagggctaat gatcgcggag 2520 aagataaaaa gtggaaaaggggcatatcct tttcattttg gtgctgcaat agctgttgta 2580 tatggtgagg ataaaaaagtagccgcttca attctgacag atttatctga acctaaaaga 2640 gacgaaggcg agtatttgcaaagtacgaga aaggtaagcg caatgtttat cacaaacgtc 2700 aatgaatttc gcggccatgattacccaaaa agtaaatata gtatcggatt agttacagct 2760 gaaaaacgtc agccagtaataagcaaaaaa cgtgcaaacc cggaagaggc cccttcatca 2820 tccagaaata aaaaattgcatgtacattaa 2850 48 2817 DNA Photorhabdus strain W14 48 atggaaaacattgacccaaa actttatcac catacgccta ccgtcagtgt tcacgataac 60 cgtggactagctatccgtaa tattagtttt caccgcacta ccgcagaagc aaataccgat 120 acccgtattacccgccatca atataatgcc ggcggatatt tgaaccaaag cattgatcct 180 cgcctgtatgacgccaaaca gactaacaac gctgtacaac cgaattttat ctggcgacat 240 aatttgaccggcaatatcct gcgaacagag agcgtcgatg ccggtcggac gattaccctc 300 aacgatattgaaggccgccc ggtgttgacc atcaatgcag ccggtgtccg gcaaaaccat 360 cgctacgaagataacaccct gcccggtcgc ctgctcgcta tcagcgaaca aggacaggca 420 gaagagaaaacgaccgagcg ccttatctgg gccggcaata cgccgcaaga aaaagaccac 480 aaccttgccggtcagtgcgt ccgccattac gataccgcag gactcactca actcaacagc 540 cttgccctgaccggcgccgt tctatcacaa tctcaacaac tgcttaccga taaccaggat 600 gccgactggacaggtgaaga ccagagcctc tggcaacaaa aactgagtag tgatgtctat 660 atcacccaaagtaacactga tgccaccggg gctttactga cccagaccga tgccaaaggc 720 aacattcagcggctggccta tgatgtggcc gggcagctaa aagggagttg gttaacactc 780 aaaggtcaggcggaacaggt gattatcaaa tcgctaacct actccgccgc cgggcaaaaa 840 ttacgtgaagagcacggtaa cgggattgtc actgaataca gctacgaacc ggaaacccaa 900 cggcttatcggcattaccac tcgccgtcca tcagacgcca aggtgttgca agacctacgc 960 tatcaatatgacccagtagg caatgtcatt agtatccgta atgatgcgga agccactcgc 1020 ttttggcgcaatcagaaagt agccccggag aatagctata cctacgattc cctgtatcag 1080 cttatcagcgccaccgggcg cgagatggcc aatatcggtc agcaaagcaa ccaacttccc 1140 tctccggcgctaccttctga taacaatacc tacaccaact atactcgcac ttatacttat 1200 gaccgtggcggcaatttgac gaaaattcag catagttcac cagccgcgca aaataactac 1260 acgacggatataacggtttc aaatcgcagc aaccgcgcgg tactcagcac attgaccgca 1320 gatccaactcaagtcgatgc cttatttgat gcgggaggcc atcaaaccag cttgttatcc 1380 ggccaagttctaacttggac accgcgaggc gaattgaaac aagccaacaa tagcgcagga 1440 aatgagtggtatcgctacga tagcaacggc atacgccagc taaaagtgaa tgaacaacaa 1500 actcagaatatcccgcaaca acaaagggta acttatctac cggggctgga aatacgtaca 1560 acccagaacaacgccacaac aacagaagag ttacacgtta tcacactcgg taaagccggc 1620 cgcgcgcaagtccgagtatt gcattgggag agcggtaaac cagaagatat taataacaat 1680 cagcttcgttacagctacga taatcttatt ggctccagcc aacttcaatt agatagcgac 1740 ggacaaattatcagtgaaga agaatattat ccatttggtg gtacagcgct gtgggcggca 1800 aggaatcaaaccgaagccag ctataaaacc attcgttatt ctggtaaaga gcgggatgtt 1860 accgggctgtattattatgg ctaccgttat taccaaccgt gggcgggcag atggttaagt 1920 gcagacccggcaggaaccat tgatggactg aatttatatc gcatggtgag aaataacccg 1980 gtgacgcaatttgatgttca gggattatca ccggccaaca gaacagaaga agcgataata 2040 aaacagggttcctttacggg aatggaagaa gctgtttata aaaaaatggc taaacctcaa 2100 actttcaaacgccaaagagc tatcgctgcc caaacagagc aagaagccca tgaatcattg 2160 accaacaaccctagtgtaga tattagccca attaaaaact acaccacaga tagctcacaa 2220 attaatgccgcgataaggga aaatcgtatt acgccagcag tggaaagttt agacgccaca 2280 ttatcttccctacaagatag acaaatgagg gtaacttatc gggtgatgac ctatgtagat 2340 aattccacgccatcgccttg gcactcgcca caggaaggaa atagtattaa tgttggtgat 2400 atcgtttcggataacgctta tttatcaaca tcggcccatc gtggttttct gaattttgtt 2460 cacaaaaaagaaaccagtga aactcgatac gtcaagatgg catttttaac gaatgcgggt 2520 gtcaatgtctcagcagcatc tatgtataat aatgctggcg aggagcaagt atttaaaatg 2580 gatttaaacgattcaagaaa aagccttgct gaaaaattaa aactaagagt cagtggacca 2640 caatcgggacaagcggaaat attactacct agggaaacac agttcgaagt tgtttcaatg 2700 aaacatcaaggcagagatac ctatgtatta ttgcaagata ttaaccaatc cgcagccact 2760 catagaaatgtacgtaacac ttacaccggt aatttcaaat catccagtgc aaattaa 2817 49 2538 PRTXenorhabdus nematophilus 49 Met Tyr Ser Thr Ala Val Leu Leu Asn Lys IleSer Pro Thr Arg Asp 1 5 10 15 Gly Gln Thr Met Thr Leu Ala Asp Leu GlnTyr Leu Ser Phe Ser Glu 20 25 30 Leu Arg Lys Ile Phe Asp Asp Gln Leu SerTrp Gly Glu Ala Arg His 35 40 45 Leu Tyr His Glu Thr Ile Glu Gln Lys LysAsn Asn Arg Leu Leu Glu 50 55 60 Ala Arg Ile Phe Thr Arg Ala Asn Pro GlnLeu Ser Gly Ala Ile Arg 65 70 75 80 Leu Gly Ile Glu Arg Asp Ser Val SerArg Ser Tyr Asp Glu Met Phe 85 90 95 Gly Ala Arg Ser Ser Ser Phe Val LysPro Gly Ser Val Ala Ser Met 100 105 110 Phe Ser Pro Ala Gly Tyr Leu ThrGlu Leu Tyr Arg Glu Ala Lys Asp 115 120 125 Leu His Phe Ser Ser Ser AlaTyr His Leu Asp Asn Arg Arg Pro Asp 130 135 140 Leu Ala Asp Leu Thr LeuSer Gln Ser Asn Met Asp Thr Glu Ile Ser 145 150 155 160 Thr Leu Thr LeuSer Asn Glu Leu Leu Leu Glu His Ile Thr Arg Lys 165 170 175 Thr Gly GlyAsp Ser Asp Ala Leu Met Glu Ser Leu Ser Thr Tyr Arg 180 185 190 Gln AlaIle Asp Thr Pro Tyr His Gln Pro Tyr Glu Thr Ile Arg Gln 195 200 205 ValIle Met Thr His Asp Ser Thr Leu Ser Ala Leu Ser Arg Asn Pro 210 215 220Glu Val Met Gly Gln Ala Glu Gly Ala Ser Leu Leu Ala Ile Leu Ala 225 230235 240 Asn Ile Ser Pro Glu Leu Tyr Asn Ile Leu Thr Glu Glu Ile Thr Glu245 250 255 Lys Asn Ala Asp Ala Leu Phe Ala Gln Asn Phe Ser Glu Asn IleThr 260 265 270 Pro Glu Asn Phe Ala Ser Gln Ser Trp Ile Ala Lys Tyr TyrGly Leu 275 280 285 Glu Leu Ser Glu Val Gln Lys Tyr Leu Gly Met Leu GlnAsn Gly Tyr 290 295 300 Ser Asp Ser Thr Ser Ala Tyr Val Asp Asn Ile SerThr Gly Leu Val 305 310 315 320 Val Asn Asn Glu Ser Lys Leu Glu Ala TyrLys Ile Thr Arg Val Lys 325 330 335 Thr Asp Asp Tyr Asp Lys Asn Ile AsnTyr Phe Asp Leu Met Tyr Glu 340 345 350 Gly Asn Asn Gln Phe Phe Ile ArgAla Asn Phe Lys Val Ser Arg Glu 355 360 365 Phe Gly Ala Thr Leu Arg LysAsn Ala Gly Pro Ser Gly Ile Val Gly 370 375 380 Ser Leu Ser Gly Pro LeuIle Ala Asn Thr Asn Phe Lys Ser Asn Tyr 385 390 395 400 Leu Ser Asn IleSer Asp Ser Glu Tyr Lys Asn Gly Val Lys Ile Tyr 405 410 415 Ala Tyr ArgTyr Thr Ser Ser Thr Ser Ala Thr Asn Gln Gly Gly Gly 420 425 430 Ile PheThr Phe Glu Ser Tyr Pro Leu Thr Ile Phe Ala Leu Lys Leu 435 440 445 AsnLys Ala Ile Arg Leu Cys Leu Thr Ser Gly Leu Ser Pro Asn Glu 450 455 460Leu Gln Thr Ile Val Arg Ser Asp Asn Ala Gln Gly Ile Ile Asn Asp 465 470475 480 Ser Val Leu Thr Lys Val Phe Tyr Thr Leu Phe Tyr Ser His Arg Tyr485 490 495 Ala Leu Ser Phe Asp Asp Ala Gln Val Leu Asn Gly Ser Val IleAsn 500 505 510 Gln Tyr Ala Asp Asp Asp Ser Val Ser His Phe Asn Arg LeuPhe Asn 515 520 525 Thr Pro Pro Leu Lys Gly Lys Ile Phe Glu Ala Asp GlyAsn Thr Val 530 535 540 Ser Ile Asp Pro Asp Glu Glu Gln Ser Thr Phe AlaArg Ser Ala Leu 545 550 555 560 Met Arg Gly Leu Gly Val Asn Ser Gly GluLeu Tyr Gln Leu Gly Lys 565 570 575 Leu Ala Gly Val Leu Asp Ala Gln AsnThr Ile Thr Leu Ser Val Phe 580 585 590 Val Ile Ser Ser Leu Tyr Arg LeuThr Leu Leu Ala Arg Val His Gln 595 600 605 Leu Thr Val Asn Glu Leu CysMet Leu Tyr Gly Leu Ser Pro Phe Asn 610 615 620 Gly Lys Thr Thr Ala SerLeu Ser Ser Gly Glu Leu Pro Arg Leu Val 625 630 635 640 Ile Trp Leu TyrGln Val Thr Gln Trp Leu Thr Glu Ala Glu Ile Thr 645 650 655 Thr Glu AlaIle Trp Leu Leu Cys Thr Pro Glu Phe Ser Gly Asn Ile 660 665 670 Ser ProGlu Ile Ser Asn Leu Leu Asn Asn Leu Arg Pro Ser Ile Ser 675 680 685 GluAsp Met Ala Gln Ser His Asn Arg Glu Leu Gln Ala Glu Ile Leu 690 695 700Ala Pro Phe Ile Ala Ala Thr Leu His Leu Ala Ser Pro Asp Met Ala 705 710715 720 Arg Tyr Ile Leu Leu Trp Thr Asp Asn Leu Arg Pro Gly Gly Leu Asp725 730 735 Ile Ala Gly Phe Met Thr Leu Val Leu Lys Glu Ser Leu Asn AlaAsn 740 745 750 Glu Thr Thr Gln Leu Val Gln Phe Cys His Val Met Ala GlnLeu Ser 755 760 765 Leu Ser Val Gln Thr Leu Arg Leu Ser Glu Ala Glu LeuSer Val Leu 770 775 780 Val Ile Ser Gly Phe Ala Val Leu Gly Ala Lys AsnGln Pro Ala Gly 785 790 795 800 Gln His Asn Ile Asp Thr Leu Phe Ser LeuTyr Arg Phe His Gln Trp 805 810 815 Ile Asn Gly Leu Gly Asn Pro Gly SerAsp Thr Leu Asp Met Leu Arg 820 825 830 Gln Gln Thr Leu Thr Ala Asp ArgLeu Ala Ser Val Met Gly Leu Asp 835 840 845 Ile Ser Met Val Thr Gln AlaMet Val Ser Ala Gly Val Asn Gln Leu 850 855 860 Gln Cys Trp Gln Asp IleAsn Thr Val Leu Gln Trp Ile Asp Val Ala 865 870 875 880 Ser Ala Leu HisThr Met Pro Ser Val Ile Arg Thr Leu Val Asn Ile 885 890 895 Arg Tyr ValThr Ala Leu Asn Lys Ala Glu Ser Asn Leu Pro Ser Trp 900 905 910 Asp GluTrp Gln Thr Leu Ala Glu Asn Met Glu Ala Gly Leu Ser Thr 915 920 925 GlnGln Ala Gln Thr Leu Ala Asp Tyr Thr Ala Glu Arg Leu Ser Ser 930 935 940Val Leu Cys Asn Trp Phe Leu Ala Asn Ile Gln Pro Glu Gly Val Ser 945 950955 960 Leu His Ser Arg Asp Asp Leu Tyr Ser Tyr Phe Leu Ile Asp Asn Gln965 970 975 Val Ser Ser Ala Ile Lys Thr Thr Arg Leu Ala Glu Ala Ile AlaGly 980 985 990 Ile Gln Leu Tyr Ile Asn Arg Ala Leu Asn Arg Ile Glu ProAsn Ala 995 1000 1005 Arg Ala Asp Val Ser Thr Arg Gln Phe Phe Thr AspTrp Thr Val 1010 1015 1020 Asn Asn Arg Tyr Ser Thr Trp Gly Gly Val SerArg Leu Val Tyr 1025 1030 1035 Tyr Pro Glu Asn Tyr Ile Asp Pro Thr GlnArg Ile Gly Gln Thr 1040 1045 1050 Arg Met Met Asp Glu Leu Leu Glu AsnIle Ser Gln Ser Lys Leu 1055 1060 1065 Ser Arg Asp Thr Val Glu Asp AlaPhe Lys Thr Tyr Leu Thr Arg 1070 1075 1080 Phe Glu Thr Val Ala Asp LeuLys Val Val Ser Ala Tyr His Asp 1085 1090 1095 Asn Val Asn Ser Asn ThrGly Leu Thr Trp Phe Val Gly Gln Thr 1100 1105 1110 Arg Glu Asn Leu ProGlu Tyr Tyr Trp Arg Asn Val Asp Ile Ser 1115 1120 1125 Arg Met Gln AlaGly Glu Leu Ala Ala Asn Ala Trp Lys Glu Trp 1130 1135 1140 Thr Lys IleAsp Thr Ala Val Asn Pro Tyr Lys Asp Ala Ile Arg 1145 1150 1155 Pro ValIle Phe Arg Glu Arg Leu His Leu Ile Trp Val Glu Lys 1160 1165 1170 GluGlu Val Ala Lys Asn Gly Thr Asp Pro Val Glu Thr Tyr Asp 1175 1180 1185Arg Phe Thr Leu Lys Leu Ala Phe Leu Arg His Asp Gly Ser Trp 1190 11951200 Ser Ala Pro Trp Ser Tyr Asp Ile Thr Thr Gln Val Glu Ala Val 12051210 1215 Thr Asp Lys Lys Pro Asp Thr Glu Arg Leu Ala Leu Ala Ala Ser1220 1225 1230 Gly Phe Gln Gly Glu Asp Thr Leu Leu Val Phe Val Tyr LysThr 1235 1240 1245 Gly Lys Ser Tyr Ser Asp Phe Gly Gly Ser Asn Lys AsnVal Ala 1250 1255 1260 Gly Met Thr Ile Tyr Gly Asp Gly Ser Phe Lys LysMet Glu Asn 1265 1270 1275 Thr Ala Leu Ser Arg Tyr Ser Gln Leu Lys AsnThr Phe Asp Ile 1280 1285 1290 Ile His Thr Gln Gly Asn Asp Leu Val ArgLys Ala Ser Tyr Arg 1295 1300 1305 Phe Ala Gln Asp Phe Glu Val Pro AlaSer Leu Asn Met Gly Ser 1310 1315 1320 Ala Ile Gly Asp Asp Ser Leu ThrVal Met Glu Asn Gly Asn Ile 1325 1330 1335 Pro Gln Ile Thr Ser Lys TyrSer Ser Asp Asn Leu Ala Ile Thr 1340 1345 1350 Leu His Asn Ala Ala PheThr Val Arg Tyr Asp Gly Ser Gly Asn 1355 1360 1365 Val Ile Arg Asn LysGln Ile Ser Ala Met Lys Leu Thr Gly Val 1370 1375 1380 Asp Gly Lys SerGln Tyr Gly Asn Ala Phe Ile Ile Ala Asn Thr 1385 1390 1395 Val Lys HisTyr Gly Gly Tyr Ser Asp Leu Gly Gly Pro Ile Thr 1400 1405 1410 Val TyrAsn Lys Thr Lys Asn Tyr Ile Ala Ser Val Gln Gly His 1415 1420 1425 LeuMet Asn Ala Asp Tyr Thr Arg Arg Leu Ile Leu Thr Pro Val 1430 1435 1440Glu Asn Asn Tyr Tyr Ala Arg Leu Phe Glu Phe Pro Phe Ser Pro 1445 14501455 Asn Thr Ile Leu Asn Thr Val Phe Thr Val Gly Ser Asn Lys Thr 14601465 1470 Ser Asp Phe Lys Lys Cys Ser Tyr Ala Val Asp Gly Asn Asn Ser1475 1480 1485 Gln Gly Phe Gln Ile Phe Ser Ser Tyr Gln Ser Ser Gly TrpLeu 1490 1495 1500 Asp Ile Asp Thr Gly Ile Asn Asn Thr Asp Ile Lys IleThr Val 1505 1510 1515 Met Ala Gly Ser Lys Thr His Thr Phe Thr Ala SerAsp His Ile 1520 1525 1530 Ala Ser Leu Pro Ala Asn Ser Phe Asp Ala MetPro Tyr Thr Phe 1535 1540 1545 Lys Pro Leu Glu Ile Asp Ala Ser Ser LeuAla Phe Thr Asn Asn 1550 1555 1560 Ile Ala Pro Leu Asp Ile Val Phe GluThr Lys Ala Lys Asp Gly 1565 1570 1575 Arg Val Leu Gly Lys Ile Lys GlnThr Leu Ser Val Lys Arg Val 1580 1585 1590 Asn Tyr Asn Pro Glu Asp IleLeu Phe Leu Arg Glu Thr His Ser 1595 1600 1605 Gly Ala Gln Tyr Met GlnLeu Gly Val Tyr Arg Ile Arg Leu Asn 1610 1615 1620 Thr Leu Leu Ala SerGln Leu Val Ser Arg Ala Asn Thr Gly Ile 1625 1630 1635 Asp Thr Ile LeuThr Met Glu Thr Gln Arg Leu Pro Glu Pro Pro 1640 1645 1650 Leu Gly GluGly Phe Phe Ala Asn Phe Val Leu Pro Lys Tyr Asp 1655 1660 1665 Pro AlaGlu His Gly Asp Glu Arg Trp Phe Lys Ile His Ile Gly 1670 1675 1680 AsnVal Gly Gly Asn Thr Gly Arg Gln Pro Tyr Tyr Ser Gly Met 1685 1690 1695Leu Ser Asp Thr Ser Glu Thr Ser Met Thr Leu Phe Val Pro Tyr 1700 17051710 Ala Glu Gly Tyr Tyr Met His Glu Gly Val Arg Leu Gly Val Gly 17151720 1725 Tyr Gln Lys Ile Thr Tyr Asp Asn Thr Trp Glu Ser Ala Phe Phe1730 1735 1740 Tyr Phe Asp Glu Thr Lys Gln Gln Phe Val Leu Ile Asn AspAla 1745 1750 1755 Asp His Asp Ser Gly Met Thr Gln Gln Gly Ile Val LysAsn Ile 1760 1765 1770 Lys Lys Tyr Lys Gly Phe Leu Asn Val Ser Ile AlaThr Gly Tyr 1775 1780 1785 Ser Ala Pro Met Asp Phe Asn Ser Ala Ser AlaLeu Tyr Tyr Trp 1790 1795 1800 Glu Leu Phe Tyr Tyr Thr Pro Met Met CysPhe Gln Arg Leu Leu 1805 1810 1815 Gln Glu Lys Gln Phe Asp Glu Ala ThrGln Trp Ile Asn Tyr Val 1820 1825 1830 Tyr Asn Pro Ala Gly Tyr Ile ValAsn Gly Glu Ile Ala Pro Trp 1835 1840 1845 Ile Trp Asn Cys Arg Pro LeuGlu Glu Thr Thr Ser Trp Asn Ala 1850 1855 1860 Asn Pro Leu Asp Ala IleAsp Pro Asp Ala Val Ala Gln Asn Asp 1865 1870 1875 Pro Met His Tyr LysIle Ala Thr Phe Met Arg Leu Leu Asp Gln 1880 1885 1890 Leu Ile Leu ArgGly Asp Met Ala Tyr Arg Glu Leu Thr Arg Asp 1895 1900 1905 Ala Leu AsnGlu Ala Lys Met Trp Tyr Val Arg Thr Leu Glu Leu 1910 1915 1920 Leu GlyAsp Glu Pro Glu Asp Tyr Gly Ser Gln Gln Trp Ala Ala 1925 1930 1935 ProSer Leu Ser Gly Ala Ala Ser Gln Thr Val Gln Ala Ala Tyr 1940 1945 1950Gln Gln Asp Leu Thr Met Leu Gly Arg Gly Gly Val Ser Lys Asn 1955 19601965 Leu Arg Thr Ala Asn Ser Leu Val Gly Leu Phe Leu Pro Glu Tyr 19701975 1980 Asn Pro Ala Leu Thr Asp Tyr Trp Gln Thr Leu Arg Leu Arg Leu1985 1990 1995 Phe Asn Leu Arg His Asn Leu Ser Ile Asp Gly Gln Pro LeuSer 2000 2005 2010 Leu Ala Ile Tyr Ala Glu Pro Thr Asp Pro Lys Ala LeuLeu Thr 2015 2020 2025 Ser Met Val Gln Ala Ser Gln Gly Gly Ser Ala ValLeu Pro Gly 2030 2035 2040 Thr Leu Ser Leu Tyr Arg Phe Pro Val Met LeuGlu Arg Thr Arg 2045 2050 2055 Asn Leu Val Ala Gln Leu Thr Gln Phe GlyThr Ser Leu Leu Ser 2060 2065 2070 Met Ala Glu His Asp Asp Ala Asp GluLeu Thr Thr Leu Leu Leu 2075 2080 2085 Gln Gln Gly Met Glu Leu Ala ThrGln Ser Ile Arg Ile Gln Gln 2090 2095 2100 Arg Thr Val Asp Glu Val AspAla Asp Ile Ala Val Leu Ala Glu 2105 2110 2115 Ser Arg Arg Ser Ala GlnAsn Arg Leu Glu Lys Tyr Gln Gln Leu 2120 2125 2130 Tyr Asp Glu Asp IleAsn His Gly Glu Gln Arg Ala Met Ser Leu 2135 2140 2145 Leu Asp Ala AlaAla Gly Gln Ser Leu Ala Gly Gln Val Leu Ser 2150 2155 2160 Ile Ala GluGly Val Ala Asp Leu Val Pro Asn Val Phe Gly Leu 2165 2170 2175 Ala CysGly Gly Ser Arg Trp Gly Ala Ala Leu Arg Ala Ser Ala 2180 2185 2190 SerVal Met Ser Leu Ser Ala Thr Ala Ser Gln Tyr Ser Ala Asp 2195 2200 2205Lys Ile Ser Arg Ser Glu Ala Tyr Arg Arg Arg Arg Gln Glu Trp 2210 22152220 Glu Ile Gln Arg Asp Asn Ala Asp Gly Glu Val Lys Gln Met Asp 22252230 2235 Ala Gln Leu Glu Ser Leu Lys Ile Arg Arg Glu Ala Ala Gln Met2240 2245 2250 Gln Val Glu Tyr Gln Glu Thr Gln Gln Ala His Thr Gln AlaGln 2255 2260 2265 Leu Glu Leu Leu Gln Arg Lys Phe Thr Asn Lys Ala LeuTyr Ser 2270 2275 2280 Trp Met Arg Gly Lys Leu Ser Ala Ile Tyr Tyr GlnPhe Phe Asp 2285 2290 2295 Leu Thr Gln Ser Phe Cys Leu Met Ala Gln GluAla Leu Arg Arg 2300 2305 2310 Glu Leu Thr Asp Asn Gly Val Thr Phe IleArg Gly Gly Ala Trp 2315 2320 2325 Asn Gly Thr Thr Ala Gly Leu Met AlaGly Glu Thr Leu Leu Leu 2330 2335 2340 Asn Leu Ala Glu Met Glu Lys ValTrp Leu Glu Arg Asp Glu Arg 2345 2350 2355 Ala Leu Glu Val Thr Arg ThrVal Ser Leu Ala Gln Phe Tyr Gln 2360 2365 2370 Ala Leu Ser Ser Asp AsnPhe Asn Leu Thr Glu Lys Leu Thr Gln 2375 2380 2385 Phe Leu Arg Glu GlyLys Gly Asn Val Gly Ala Ser Gly Asn Glu 2390 2395 2400 Leu Lys Leu SerAsn Arg Gln Ile Glu Ala Ser Val Arg Leu Ser 2405 2410 2415 Asp Leu LysIle Phe Ser Asp Tyr Pro Glu Ser Leu Gly Asn Thr 2420 2425 2430 Arg GlnLeu Lys Gln Val Ser Val Thr Leu Pro Ala Leu Val Gly 2435 2440 2445 ProTyr Glu Asp Ile Arg Ala Val Leu Asn Tyr Gly Gly Ser Ile 2450 2455 2460Val Met Pro Arg Gly Cys Ser Ala Ile Ala Leu Ser His Gly Val 2465 24702475 Asn Asp Ser Gly Gln Phe Met Leu Asp Phe Asn Asp Ser Arg Tyr 24802485 2490 Leu Pro Phe Glu Gly Ile Ser Val Asn Asp Ser Gly Ser Leu Thr2495 2500 2505 Leu Ser Phe Pro Asp Ala Thr Asp Arg Gln Lys Ala Leu LeuGlu 2510 2515 2520 Ser Leu Ser Asp Ile Ile Leu His Ile Arg Tyr Thr IleArg Ser 2525 2530 2535

1. A method of screening a culture of a Paenibacillus isolate for a genethat encodes a protein selected from the group consisting of a Cryprotein that is toxic to a lepidopteran pest and a toxin complexprotein, wherein said method comprises at least one of the followingsteps: (a) obtaining DNA from said culture and assaying said DNA for thepresence of said gene; and (b) obtaining protein produced by saidculture and assaying said protein for the presence of a protein thatindicates the presence of said gene in said isolate.
 2. A method ofscreening a culture of a Paenibacillus isolate for a protein that hastoxin activity against a lepidopteran pest wherein said method comprisesat least one of the following steps: (a) obtaining culture brothproduced by said culture and assaying said broth for toxin activityagainst a lepidopteran pest; and (b) feeding a plurality of saidisolates to a lepidopteran pest and observing said pest for effects of atoxin.
 3. The method of claim 1 wherein said method comprises screeninga collection of Paenibacillus isolates for said protein, and saidisolate is in said collection.
 4. The method of claim 1 wherein saidprotein is a toxin complex protein.
 5. The method of claim 4 whereinsaid protein enhances the activity of a toxin complex toxin protein. 6.The method of claim 1 wherein said protein is a Cry protein that istoxic to a lepidopteran pest.
 7. The method of claim 1 wherein said stepof obtaining DNA from said culture comprises creating a library ofclones from said DNA and assaying at least one of said clones for thepresence of said gene.
 8. The method of claim 7 wherein said step ofassaying said clone for the presence of said polynucleotide comprisesassaying said clone for lepidopteran toxin activity, thereby indicatingthe presence of said polynucleotide.
 9. The method of claim 1 whereinsaid step of assaying said DNA comprises performing polymerase chainreaction with at least one primer that is designed to indicate thepresence of said gene.
 10. The method of claim 1 wherein said step ofassaying said protein comprises immunoreacting an antibody with saidprotein wherein said antibody is designed to indicate the presence ofsaid protein.
 11. The method of claim 1 wherein said step of assayingsaid DNA comprises hybridizing a nucleic acid probe to said DNA whereinsaid probe is designed to indicate the presence of said gene.
 12. Anisolated protein that has toxin activity against an insect pest whereinsaid protein is encoded by a polynucleotide sequence that hybridizeswith the complement of a sequence selected from the group consisting ofSEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 32, 34, 35, 38, and
 40. 13. Theprotein of claim 12 wherein said protein is a Cry protein and said probeis the complement of SEQ ID NO:14.
 14. The protein of claim 12 whereinsaid protein is a toxin complex protein and said probe is the complementof a sequence selected from the group consisting of SEQ ID NOS:2, 4, 6,8, 10, 12, 32, 34, 35, 38, and
 40. 15. An immunoreactive fragment of aprotein according to claim
 12. 16. An isolated polynucleotide thatencodes a protein according to claim
 12. 17. A cell comprising apolynucleotide according to claim
 16. 18. The cell according to claim 17wherein said cell is selected from the group consisting of a plant celland a microbial cell.
 19. A method of controlling an insect pest whereinsaid method comprises the step of contacting said pest with a proteincomprising an amino acid sequence selected from the group consisting ofSEQ ID NOS:3, 5, 7, 9, 11, 13, 15, 18, 19, 33, 36, 37, 39, and
 41. 20.The method of claim 1 wherein said Paenibacillus isolate is of a speciesselected from the group consisting of P. apiarius, P. chondroitinus, P.alginolyticus, P. larvae, P. validus, P. gordonae, P. alvei, P.lentimorbus, P. popilliae, P. thiaminolyticus, P. curdlanolyticus, P.kobensis, P. glucanolyticus, P. lautus, P. chibensis, P. macquariensis,P. aztofixans, P. peoriae, P. polymyxa, P. illinoisensis, P.amylolyticus, P. pabuli, and P. macerans.
 21. The method of claim 11wherein said probe is derived from a gene selected from the groupconsisting of tcaA, tcaB, tcaC, tcbA, tccA, tccB, tccC, tcdA, tcdB,xptA1, xptD1, xptB1, xptC1, xptA2, sepA, sepB, and sepC.
 22. The methodof claim 9 wherein said primer is derived from a gene selected from thegroup consisting of tcaA, tcaB, tcaC, tcbA, tccA, tccB, tccC, tcdA,tcdB, xptA1, xptD1, xptB1, xptC1, xptA2, sepA, sepB, and sepC.
 23. Themethod of claim 9 wherein said primer is selected from the groupconsisting of SEQ ID NOS:22, 23, 24, 25, 26, 27, 28, 29, 30, and
 31. 24.A biologically pure culture of a Paenibacillus strain selected from thegroup consisting of DAS1529 (available under NRRL B-30599) and DB482(available under NRRL B-30670).